Repeat protein from collection of repeat proteins comprising repeat modules

ABSTRACT

A repeat protein from a collection of repeat proteins, wherein each repeat protein of said collection comprises a repeat domain, which comprises a set of consecutive repeat modules, wherein the repeat modules have the same fold and stack tightly to create a superhelical structure having a joint hydrophobic core, wherein each of the repeat modules is derived from one or more repeat units and wherein the repeat units comprise framework residues, which contribute to the folding topology of the repeat unit or contribute to an interaction with a neighboring repeat unit, and target interaction residues, which contribute to an interaction with a target substance, wherein the repeat proteins of the collection differ from other repeat proteins in the collection in at least one amino acid position of the repeat modules is described as are related pharmaceuticals and nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATION

This is a Divisional Application of U.S. patent application Ser. No.13/330,336, filed Dec. 19, 2011, which is a Divisional Application ofU.S. patent application Ser. No. 12/222,525, filed Aug. 11, 2008, nowU.S. Pat. No. 8,110,653, issued Feb. 7, 2012; which is a DivisionalApplication of U.S. patent application Ser. No. 10/363,552, filed Jul.6, 2003, now U.S. Pat. No. 7,417,130, issued Aug. 26, 2008; which is aNational Stage entry of International Application No. PCT/EP2001/010454,filed Sep. 10, 2001. The disclosures of the prior applications arehereby incorporated in their entirety by reference.

The present invention relates to collections of repeat proteinscomprising repeat modules which are derived from one or more repeatunits of a family of naturally occurring repeat proteins, to collectionsof nucleic acid molecules encoding said repeat proteins, to methods forthe construction and application of such collections and to individualmembers of such collections.

A number of documents are cited throughout this specification. Thedisclosure content of these documents is herewith incorporated byreference.

Protein-protein interactions, or more generally, protein-ligandinteractions, play an important role in all organisms and theunderstanding of the key features of recognition and binding is onefocus of current biochemical research. Up to now, antibodies and any ofthe derivatives, which have been elaborated, are mainly used in thisfield of research. However, antibody technology is afflicted withwell-known disadvantages. For instance, antibodies can hardly be appliedintracellularly due to the reductive environment in the cytoplasm. Thus,there exists a need for high affinity binding molecules withcharacteristics that overcome the restriction of antibodies. Suchmolecules will most probably provide new solutions in medicine,biotechnology, and research, where intracellular binders will alsobecome increasingly important in genomics.

Various efforts to construct novel binding proteins have been reported(reviewed in Nygren and Uhlen, 1997). The most promising strategy seemedto be a combination of limited library generation and screening orselection for the desired properties. Usually, existing scaffolds wererecruited to randomise some exposed amino acid residues after analysisof the crystal structure. However, despite progress in terms ofstability and expressibility, the affinities reported so far areconsiderably lower than the ones of antibodies (Ku and Schultz, 1995). Aconstraint might be the limitation to targets for which the crystalstructure is known (Kirkham et al., 1999) or which are homologous to theoriginal target molecule, so that no universal scaffold for binding hasbeen identified so far. To increase the apparent affinity of bindersafter screening, several approaches have used multimerisation of singlebinders to take advantage of avidity effects.

Thus, the technical problem underlying the present invention is toidentify novel approaches for the construction of collections of bindingproteins.

The solution to this technical problem is achieved by providing theembodiments characterised in the claims. Accordingly, the presentinvention allows constructing collections of repeat proteins comprisingrepeat modules. The technical approach of the present invention, i.e. toderive said modules from the repeat units of naturally occurring repeatproteins, is neither provided nor suggested by the prior art.

Thus, the present invention relates to collections of nucleic acidmolecules encoding collections of repeat proteins, each repeat proteincomprising a repeat domain, which comprises a set of consecutive repeatmodules, wherein each of said repeat modules is derived from one or morerepeat units of one family of naturally occurring repeat proteins,wherein said repeat units comprise framework residues and targetinteraction residues, wherein said repeat proteins differ in at leastone position corresponding to one of said target interaction residues.

In the context of the present invention, the term “collection” refers toa population comprising at least two different entities or members.Preferably, such a collection comprises at least 10⁵, more preferablymore than 10⁷, and most preferably more that 10⁹ different members. A“collection” may as well be referred to as a “library” or a “plurality”.

The term “nucleic acid molecule” refers to a polynucleotide molecule,which is a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)molecule, either single, stranded or double stranded. A nucleic acidmolecule may either be present in isolated form, or be comprised inrecombinant nucleic acid molecules or vectors. The term “repeat protein”refers to a (poly)peptide/protein comprising one or more repeat domains(FIG. 1). Preferably, each of said repeat proteins comprises up to fourrepeat domains. More preferably, each of said repeat proteins comprisesup to two repeat domains. However, most preferably, each of the repeatproteins comprises one repeat domain. Furthermore, said repeat proteinmay comprise additional non-repeat protein domains (FIGS. 2a and 2b ),(poly)peptide tags and/or (poly)peptide linker sequences (FIG. 1). Theterm “(poly)peptide tag” refers to an amino acid sequence attached to a(poly)peptide/protein, where said amino acid sequence is usable for thepurification, detection, or targeting of said (poly)peptide/protein, orwhere said amino acid sequence improves the physio-chemical behavior ofsaid (poly)peptide/protein, or where said amino acid sequence possessesan effector function. Such (poly)peptide tags may be small polypeptidesequences, for example, His_(n) (Hochuli et al., 1988; Lindner et al.,1992), myc, FLAG (Hopp et al., 1988; Knappik and Plückthun, 1994), orStrep-tag (Schmidt and Skerra, 1993; Schmidt and Skerra, 1994; Schmidtet al., 1996. These (poly)peptide tags are all well known in the art andare fully available to the person skilled in the art. Additionalnon-repeat domains may be further moieties such as enzymes (for exampleenzymes like alkaline phosphatase), which allow the detection of saidrepeat proteins, or moieties which can be used for targeting (such asimmunoglobulins or fragments thereof) and/or as effector molecules. Theindividual (poly)peptide tags, moieties and/or domains of a repeatprotein may be connected to each other directly or via (poly)peptidelinkers. The term “(poly)peptide linker” refers to an amino acidsequence, which is able to link, for example two protein domains, a(poly)peptide tag and a protein domain or two sequence tags. Suchlinkers for example glycine linkers of variable lengths (e.g. Forrer andJaussi, 1998), are known to the person skilled in the relevant art.

In the context of the present invention, the term “(poly)peptide”relates to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds.

The term “protein” refers to a (poly)peptide, where at least part of the(poly)peptide has, or is able to, acquire a defined three-dimensionalarrangement by forming secondary, tertiary, or quaternary structureswithin and/or between its (poly)peptide chain(s). If a protein comprisestwo or more (poly)peptides, the individual (poly)peptide chains may belinked non-covalently or covalently, e.g. by a disulfide bond betweentwo (poly)peptides. A part of a protein, which individually has, or isable to, acquire a defined three-dimensional arrangement by formingsecondary or tertiary structures is termed “protein domain”. Suchprotein domains are well known to the practitioner skilled in therelevant art.

The term “family of naturally occurring repeat proteins” refers to agroup of naturally occurring repeat proteins, where the members of saidgroup comprise similar repeat units. Protein families are well known tothe person skilled in the art.

The term “repeat domain” refers to a protein domain comprising two ormore consecutive repeat units (modules) as structural units (FIG. 1),wherein said structural units have the same fold, and stack tightly tocreate a superhelical structure having a joint hydrophobic core (for areview see Kobe and Kajava, 2000). The term “structural unit” refers toa locally ordered part of a (poly)peptide, formed by three-dimensionalinteractions between two or more segments of secondary structure thatare near one another along the (poly)peptide chain. Such a structuralunit comprises a structural motif. The term “structural motif” refers toa three-dimensional arrangement of secondary structure elements presentin at least one structural unit. For example, the structural motifrepetitively present in LRR proteins consists of a β-strand and anopposing antiparallel helical segment connected by a loop (FIG. 4a ).Structural motifs are well known to the person skilled in the relevantart. Said structural units are alone not able to acquire a definedthree-dimensional arrangement; however, their consecutive arrangement asrepeat modules in a repeat domain leads to a mutual stabilization ofneighbouring units resulting in said superhelical structure.

The term “repeat modules” refers to the repeated amino acid sequences ofthe repeat proteins encoded by the nucleic acid molecules of thecollection of the present invention, which are derived from the repeatunits (FIG. 3) of naturally occurring proteins. Each repeat modulecomprised in a repeat domain is derived from one or more repeat units ofone family of naturally occurring repeat proteins.

Such “repeat modules” may comprise positions with amino acid residuespresent in all copies of the repeat module (“fixed positions”) andpositions with differing or “randomised” amino acid residues(“randomised positions”).

The term “set of repeat modules” refers to the total number of repeatmodules present in a repeat domain. Such “set of repeat modules” presentin a repeat domain comprises two or more consecutive repeat modules, andmay comprise just one type of repeat module in two or more copies, ortwo or more different types of modules, each present in one or morecopies. The collection of repeat proteins according to the presentinvention may comprise repeat domains with identical number of repeatmodules per corresponding repeat domain (i.e. one set with a fixednumber of repeat modules), or may comprise repeat domains, which differin the number of repeal modules per corresponding repeat domain (i.e.two or more sets with different numbers of repeat modules).

Preferably, the repeat modules comprised in a set are homologous repeatmodules. In the context of the present invention, the term “homologousrepeat modules” refers to repeat modules, wherein more than 70% of theframework residues of said repeat modules are homologous. Preferably,more than 80% of the framework residues of said repeat modules arehomologous. Most preferably, more than 90% of the framework residues ofsaid repeat modules are homologous. Computer programs to determine thepercentage of homology between polypeptides, such as Fasta, Blast orGap, are known to the person skilled in the relevant art.

Preferably, a repeat module of the present invention is derived from onerepeat unit. This may refer to a situation where a collection of nucleicacid molecules, each molecule encoding a repeat domain of the invention,is obtained by random mutagenesis of a nucleic acid molecule encoding anaturally occurring repeat domain. Thus, said repeat domain of thepresent invention comprises a set of repeat modules, wherein each ofsaid modules is derived from the corresponding repeat unit of saidnaturally occurring repeat domain. Methods for random mutagenesis ofnucleic acid molecules such as error-prone PCR (Wilson and Keefe, 2000)or DNA shuffling (Volkov and Arnold, 2000) are well known to the personskilled in the relevant art. In another situation, a single naturallyoccurring repeat unit may be used to derive a repeat sequence motif ofthe present invention.

More preferably, a repeat module of the present invention is derivedfrom one or more repeat units. This may refer to a situation where twoor more homologous nucleic acid molecules, each encoding a naturallyoccurring repeat domain, are subjected to DNA recombination or randomchimeragenesis (Volkov and Arnold, 2000). Thus, said repeat domain ofthe present invention comprises a set of repeat modules, wherein each ofsaid modules is derived from one or more corresponding repeat units ofsaid homologous naturally occurring repeat domains. Preferably, saidhomologous nucleic acid molecules possess a DNA sequences identity of atleast 75%. More preferably said sequence identity is at least 85%.

Most preferably, a repeat module of the present invention is derivedfrom two or more repeat units, where two or more homologous repeat unitsare used to derive a repeat sequence motif of the present invention.Descriptions of such a derivation process are presented in the examples.

The term “a repeat module derived from one or more repeat units” refersto

-   -   (i) a process comprising the analysis of one or more repeat        units of naturally occurring repeat proteins and the deduction        of a repeat module. This process may comprise the steps of:        -   (a) identifying naturally occurring repeat units;        -   (b) determining an initial repeat sequence motif by sequence            alignments;        -   (c) refining the repeat sequence motif by sequence analysis            and structural analysis of said repeat units;        -   (d) constructing a repeat module according to the repeat            sequence motif of (c); or    -   (ii) a process comprising the process of (i) followed by further        evolution of the repeat module by random mutagenesis or random        chimeragenesis.

The term “repeat unit” refers to amino acid sequences comprisingsequence motifs of one or more naturally occurring proteins, whereinsaid “repeat units” are found in multiple copies, and which exhibit adefined folding topology common to all said motifs determining the foldof the protein. Such repeat units comprise framework residues (FIG. 4d )and interaction residues (FIG. 4e ). Examples of such repeat unitsinclude leucine-rich repeat units, ankyrin repeat units, armadillorepeat units, tetratricopeptide repeat units. HEAT repeat units, andleucine-rich variant repeat units (reviewed in Kobe & Deisenhofer, 1994;Groves & Barford, 1999; Marino et al., 2000; Kobe, 1996). Naturallyoccurring proteins containing two or more such repeat units are referredto as “naturally occurring repeat proteins”. The amino acid sequences ofthe individual repeat units of a repeat protein may have a significantnumber of mutations, substitutions, additions and/or deletions whencompared to each other, while still substantially retaining the generalpattern, or motif, of the repeat units.

Preferably, the repeat units used for the deduction of a repeat sequencemotif are homologous repeat units, wherein the repeat units comprise thesame structural motif and wherein more than 70% of the frameworkresidues of said repeat units are homologous. Preferably, more than 80%of the framework residues of said repeat units are homologous. Mostpreferably, more than 90% of the framework residues of said repeat unitsare homologous.

The term “repeat sequence motif” refers to an amino acid sequence, whichis deduced from one or more repeat units. Such repeat sequence motifscomprise framework residue positions and target interaction residuepositions. Said framework residue positions correspond to the positionsof framework residues of said repeat units. Said target interactionresidue positions correspond to the positions of target interactionresidues of said repeat units. Such repeat sequence motifs comprisefixed positions and randomized positions. The term “fixed position”refers to an amino acid position in a repeat sequence motif, whereinsaid position is set to a particular amino acid. Most often, such fixedpositions correspond to the positions of framework residues. The term“randomized position” refers to an amino acid position in a repeatsequence motif, wherein two or more amino acids are allowed at saidamino acid position. Most often, such randomized positions correspond tothe positions of target target interaction residues. However, somepositions of framework residues may also be randomized. Amino acidsequence motifs are well known to the practitioner in the relevant art.

The term “folding topology” refers to the tertiary structure of saidrepeat units. The folding topology will be determined by stretches ofamino acids forming at least parts of α-helices or β-sheets, or aminoacid stretches forming linear polypeptides or loops, or any combinationof α-helices, β-sheets and/or linear polypeptides/loops. The term“consecutive” refers to an arrangement, wherein said modules arearranged in tandem.

In repeat proteins, there are at least 2, usually about 2 to 6, moreusually at least about 6, frequently 20 or more repeat units. For themost part, the repeat proteins are structural proteins and/or adhesiveproteins, being present in prokaryotes and eukaryotes, includingvertebrates and non-vertebrates. An analogy of ankyrin proteins toantibodies has been suggested (Jacobs and Harrison, 1998).

In most cases, said repeat units will exhibit a high degree of sequenceidentity (same amino acid residues at corresponding positions) orsequence similarity (amino acid residues being different, but havingsimilar physicochemical properties), and some of the amino acid residuesmight be key residues being strongly conserved in the different repeatunits found in naturally occurring proteins.

However, a high degree of sequence variability by amino acid insertionsand/or deletions, and/or substitutions between the different repeatunits found in naturally occurring proteins will be possible as long asthe common folding topology is maintained.

Methods for directly determining the folding topology of repeat proteinsby physicochemical means such as X-ray crystallography, NMR or CDspectroscopy, are well known to the practitioner skilled in the relevantart. Methods for identifying and determining repeat units or repeatsequence motifs, or for identifying families of related proteinscomprising such repeat units or motifs, such as homology searches (BLASTetc.) are well established in the field of bioinformatics, and are wellknown to the practitioner in such art. The step of refining an initialrepeat sequence motif may comprise an iterative process.

Crystal structures have been reported for ankyrin-type repeats (Bork,1993; Huxford et al., 1998, see FIGS. 2g and 2h ), the ribonucleaseinhibitor (RI) of the leucine-rich repeat (LRR) superfamily (Kobe andDeisenhofer, 1993, see FIG. 2c ) and other LRR proteins (see FIGS. 2d to2f ). Inspection of these structures revealed an elongated shape in thecase of the ankyrin repeats, or a horseshoe shape in the case of theleucine-rich repeats giving rise to an extraordinarily large surface.

The term “framework residues” relates to amino acid residues of therepeat units, or the corresponding amino acid residues of the repeatmodules, which contribute to the folding topology, in which contributeto the fold of said repeat unit (or module) or which contribute to theinteraction with a neighboring unit (or module). Such contribution mightbe the interaction with other residues in the repeat unit (module) (4d),or the influence on the polypeptide backbone conformation as found inα-helices or β-sheets, or amino acid stretches forming linearpolypeptides or loops. The term “target interaction residues” refers toamino acid residues of the repeat units or the corresponding amino acidresidues of the repeat modules, which contribute to the interaction withtarget substances. Such contribution might be the direct interactionwith the target substances (FIG. 4e ), or the influence on otherdirectly interacting residues, e.g. by stabilising the conformation ofthe (poly)peptide of said repeat unit (module) to allow or enhance theinteraction of said directly interacting residues with said target. Suchframework and target interaction residues may be identified by analysisof the structural data obtained by the physicochemical methods referredto above, or by comparison with known and related structural informationwell known to practitioners in structural biology and/or bioinformatics.

The term “interaction with said target substances” may be, without beinglimited to, binding to a target, involvement in a conformational changeor a chemical reaction of said target, or activation of said target.

A “target” may be an individual molecule such as a nucleic acidmolecule, a (poly)peptide or protein, a carbohydrate, or any othernaturally occurring molecule, including any part of such individualmolecule, or complexes of two or more of such molecules. The target maybe a whole cell or a tissue sample, or it may be any non-naturallyoccurring molecule or moiety.

The term “differ in at least one position” refers to a collection ofrepeat proteins, which have at least one position where more than oneamino acid may be found. Preferably, such positions are randomised. Theterm “randomised” refers to positions of the repeat modules, which arevariable within a collection and are occupied by more than one aminoacid residue in the collection. Preferably, the randomised positionsvary additionally between repeat modules within one repeat domain.Preferably, such positions may be fully randomised, i.e., being occupiedby the full set of naturally occurring, proteinogenic amino acidresidues. More preferably, such positions may be partially randomised,i.e. being occupied by a subset of the full set of naturally occurringamino acid residues. Subsets of amino acid residues may be sets of aminoacid residues with common physicochemical properties, such as sets ofhydrophobic, hydrophilic, acidic, basic, aromatic, or aliphatic aminoacids, subsets comprising all except for certain non-desired amino acidresidues, such as sets not comprising cysteines or prolines, or subsetscomprising all amino acid residues found at the corresponding positionin naturally occurring repeat proteins. The randomisation may be appliedto some, preferably to all of the target interaction residues. Methodsfor making “randomised” repeat proteins such as by usingoligonucleotide-directed mutagenesis of the nucleic acid sequencesencoding said repeat proteins (e.g. by using mixtures of mononucleotidesor trinucleotides (Virnekäs et al., 1994)), or by using error-prone FORduring synthesis of said nucleic acid sequences, are well known to thepractitioner skilled in the art.

In a preferred embodiment, each of said repeat modules has an amino acidsequence, wherein at least 70% of the amino acid residues correspondeither

-   -   (i) to consensus amino acid residues deduced from the amino acid        residues found at the corresponding positions of at least two        naturally occurring repeat units; or    -   (ii) to the amino acid residues found at the corresponding        positions in a naturally occurring repeat unit.

A “consensus amino acid residue” may be found by aligning two or morerepeat units based on structural and/or sequence homology determined asdescribed above, and by identifying one of the most frequent amino acidresidue for each position in said units (an example is shown in FIGS. 5aand 5b ). Said two or more repeat units may be taken from the repeatunits comprised in a single repeat protein, or from two or more repeatproteins. If two or more amino acid residues are found with a similarprobability in said two ore more repeat units, the consensus amino acidmay be one of the most frequently found amino acid or a combination ofsaid two or more amino acid residues.

Further preferred is a collection, wherein said set consists of betweentwo and about 30 repeat modules.

More preferably, said set consists of between 6 and about 15 repeatmodules.

In a yet further preferred embodiment of the present invention, saidrepeat modules are directly connected.

In the context of the present invention, the term “directly connected”refers to repeat modules, which are arranged as direct repeats in arepeat protein without an intervening amino acid sequence.

In a still further preferred embodiment, said repeat modules areconnected by a (poly)peptide linker.

Thus, the repeat modules may be linked indirectly via a (poly)peptidelinker as intervening sequence separating the individual modules. An“intervening sequence” may be any amino acid sequence, which allows toconnect the individual modules without interfering with the foldingtopology or the stacking of the modules. Preferentially, saidintervening sequences are short (poly)peptide linkers of less than 10,and even more preferably, of less than 5 amino acid residues.

In a still further preferred embodiment of the collection of the presentinvention, each of said repeat proteins further comprises an N- and/or aC-terminal capping module (FIG. 1) having an amino acid sequencedifferent from any one of said repeat modules.

The term “capping module” refers to a polypeptide fused to the N- orC-terminal repeat module of a repeat domain, wherein said capping moduleforms tight tertiary interactions with said repeat module therebyproviding a cap that shields the hydrophobic core of said repeat moduleat the side not in contact with the consecutive repeat module from thesolvent (FIG. 1).

Said N- and/or C-terminal capping module may be, or may be derived from,a capping unit (FIG. 3) or other domain found in a naturally occurringrepeat protein adjacent to a repeat unit. The term “capping unit” refersto a naturally occurring folded (poly)peptide, wherein said(poly)peptide defines a particular structural unit which is N- orC-terminally fused to a repeat unit, wherein said (poly)peptide formstight tertiary interactions with said repeat unit thereby providing acap that shields the hydrophobic core of said repeat unit at one sidefrom the solvent. Such capping units may have sequence similarities tosaid repeat sequence motif.

In a preferred embodiment, the present invention relates to a collectionof nucleic acid molecules, wherein said repeat units are ankyrin repeatunits.

The characteristics of ankyrin repeat proteins have been reviewed(Sedgwick and Smerdon, 1990) and one minimal folding unit has beeninvestigated (Zhang and Peng, 2000). Ankyrin repeat proteins have beenstudied in some detail, and the data can be used to exemplify theconstruction of repeat proteins according to the present invention.

Ankyrin repeat proteins have been identified in 1987 through sequencecomparisons between four such proteins in Saccharomyces cerevisiae,Drosophila melanogaster and Caenorhabditis elegans. Breeden and Nasmythreported multiple copies of a repeat unit or approximately 33 residuesin the sequences of swi6p, cdc10p, notch and lin-12 (Breeden andNasmyth, 1987). The subsequent discovery of 24 copies of this repeatunit in the ankyrin protein led to the naming of this repeat unit as theankyrin repeat (Lux et al., 1990). Later, this repeat unit has beenidentified in several hundreds of proteins of different organisms andviruses (Bork, 1993; SMART database, Schultz et al., 2000). Theseproteins are located in the nucleus, the cytoplasm or the extracellularspace. This is consistent with the fact that the ankyrin repeat domainof these proteins is independent of disulfide bridges and thusindependent of the oxidation state of the environment. The number ofrepeat units per protein varies from two to more than twenty (SMARTdatabase, Schultz et al., 2000). A minimum number of repeat units seemsto be required to form a stable folded domain (Zhang and Peng, 2000). Onthe other hand, there is also some evidence for an upper limit of sixrepeat units being present in one folded domain (Michaely and Bennet,1993).

All so far determined tertiary structures of ankyrin repeat units sharea characteristic fold (Sedgwick and Smerdon, 1999) composed of aβ-hairpin followed by two antiparallel α-helices and ending with a loopconnecting the repeat unit with the next one (FIG. 4c ). Domains builtof ankyrin repeat units are formed by stacking the repeat units to anextended and curved structure. This is illustrated by the structure ofthe mouse GA-binding protein beta 1 subunit in FIG. 2 h.

Proteins containing ankyrin repeat domains often contain additionaldomains (SMART database, Schultz et al., 2000). While the latter domainshave variable functions, the function of the ankyrin repeat domain ismost often the binding of other proteins, as several examples show(Batchelor et al., 1998; Gonna and Pavletich, 1996; Huxford et al.,1999; Jacobs and Harrisson, 1999; Jeffrey et al., 2000). When analysingthe repeat units of these proteins, the target interaction residues aremainly found in the β-hairpin and the exposed part of the first α-helix(FIG. 4c ). These target interaction residues are hence forming alargecontact surface on the ankyrin repeat domain. This contact surfaceis exposed on a framework built of stacked units of α-helix 1, α-helix 2and the loop (FIG. 4c ). For an ankyrin repeat protein consisting offive repeat units, this interaction surface contacting other proteins isapproximately 1200 Å². Such a large interaction surface is advantageousto achieve high affinities to target molecules. The affinity of IkBa(which contains a domain of six ankyrin repeat units) to the NF-kBheterodimer for example is K_(D)=3 nM (Malek at al., 1998), whereas thedissociation constant of human GA-binding protein beta 1 to its alphaunit is K_(D)=0.78 nM (Suzuki et al., 1998 An advantage of the use ofankyrin repeat proteins according to the present invention over widelyused antibodies is their potential to be expressed in a recombinantfashion in large amounts as soluble, monomeric and stable molecules(example 2).

Further preferred is a collection, wherein each of said repeat modulescomprises the ankyrin repeat consensus sequence

-   -   DxxGxTPLHLAaxx±±±±±±±±±±GpxpaVpxLLpxGA±±±±±DVNAx (SEQ ID NO: 1),        wherein “x” denotes any amino acid, “±” denotes any amino acid        or a deletion, “a” denotes an amino acid with an a polar side        chain, and “p” denotes a residue with a polar sidechain. Most        preferred is a collection, wherein one or more of the positions        denoted “x” are randomised.

Particularly preferred is a collection, wherein each of said repeatmodules comprises the ankyrin repeat consensus sequence

-   -   DxxGxTPLHLAxxxGxxxVVxLLLxxGADVNAx (SEQ ID NO: 2),        wherein “x” denotes any amino acid.

Even more preferred is a diverse collection, wherein each of said repeatmodules comprises the ankyrin repeat sequence motif

-   -   DxxGxTPLHLAxxxGxxxIVxVLLxxGADVNAx (SEQ ID NO: 3),        wherein “x” denotes any amino acid.

Yet more preferred is a diverse collection, wherein each of said repeatmodules comprises the ankyrin repeat sequence motif

-   -   D11G1TPLHLAA11GHLEIVEVLLK2GADVNA1 (SEQ ID NO: 4),        wherein 1 represents an amino acid residue selected from the        group:

A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y;

wherein 2 represents an amino acid residue selected from the group:

H, N and Y.

In a further preferred embodiment, the present invention relates to acollection of nucleic acid molecules, wherein said repeat units areleucine-rich repeats (LRR).

The characteristics and properties of the LRR repeat have been reviewed(Kobe and Deisenhofer, 1994). LRR proteins have been studied in somedetail, and the data can be used to exemplify the behaviour of repeatproteins.

LRR proteins have been identified by their highly conserved consensus ofleucine or other hydrophobic residues at positions 2, 5, 7, and 12 (FIG.4b ). However, the significance of this amino acid distribution patternwas only understood, when the first structure of an LRR, theribonuclease inhibitor protein was solved (FIG. 2c ). Recently, furtherLRR crystal structures have been elucidated (FIG. 2d-2f ). A Structureof a typical ankyrin repeat domain protein is shown for comparison (FIG.2g ). A single LRR is postulated to always correspond to a β-strand andan antiparallel α-helix (a unique α/β fold. FIG. 4a ), surrounding acore made up from leucine or other aliphatic residues only (Kajava,1998). The overall shape of ribonuclease inhibitor (RI), a LRR protein,could be described as a horseshoe (FIG. 2c ) formed by 15 tandemhomologous repeats of strictly alternating A-type (29 amino acids) andB-type (28 amino acids) LRR. The alternating nature of the protein wasalready recognised when the sequence was analysed (FIG. 5a , (Lee etal., 1988)).

Interestingly, mammalian RI are characterised by their extreme affinityto their target proteins. For the binding of RNase A to human RI aK_(i)=5.9×10⁻¹⁴ M (Kobe and Deisenhofer, 1996) was reported, whereasangiogenin was found to be inhibited with K_(i)=7.1×10⁻¹⁶ M by pig RI(Lee et al., 1989), thus becoming one of the strongest interaction knownbetween proteins. Even the best-binding antibodies feature affinitiesonly up 1.5×10⁻¹¹ M (Yang et al., 1995). To better understand theoutstanding affinity, two RI were co-crystallised with their targetproteins. Subsequent analysis of the crystal structures showed that theinteractions are mainly electrostatic (Kobe and Deisenhofer, 1996) andthe involved amino acids were predominantly found emanating from theinner β-sheet and the loop connecting each unit to its α-helix (FIG. 4b, Kobe and Deisenhofer, 1995). Moreover, the width of the horseshoe-likefold can change slightly to accommodate the target protein (Kobe andDeisenhofer, 1994). The interface between target and inhibitor consistsof a “patch-work” of interactions and the tight association originatesfrom the large buried surface area (about 2550 A²) when the targetprotein is bound inside the horseshoe, rather than shape complementarity(Kobe and Deisenhofer, 1996).

When comparing the detailed binding of RNase A and angiogenin (twomolecules with only 30% sequence identity) to RI, significantdifferences became apparent (Chen and Shapiro, 1997). Whereas largelythe same residues were involved on the side of RI, the residues of thetarget protein were not homologous or used different types of bonding(Papageorgiou et al., 1997). In other words, RI evolved in a way whichallowed it to bind and inhibit different target molecules by relying ona large number of contacts presented in correct geometrical orientation,rather than optimal complementarity of the residues. This is the basisfor a design of new binding molecules, which will have new bindingspecificities. The shape seems to be predestined for the recognition oflarge surfaces thereby allowing a much greater variety of random aminoacids to generate a library as compared to the relatively small“variable” domains of antibodies. However, the loops of antibodies seemto be superior if small haptens or deep clefts have to be recognised. Inaddition, not only the repeats themselves can be varied but also theirnumber depending on the target molecules.

Further preferred is a collection, wherein each of said modulescomprises the LRR consensus sequence

-   -   xLxxLxLxxN±xaxx±a±±±±a±±a±±x±± (SEQ ID NO: 5),        wherein “x” denotes any amino acid, “a” denotes an aliphatic        amino acid, and “±” denotes any amino acid or a deletion.

The term “aliphatic amino acid” refers to an amino acid taken from thelist of Ala, Gly, Ile, Leu, and Val.

Particularly preferred is a collection, wherein at least one of saidmodules comprises the LRR consensus sequence

-   -   xLExLxLxxCxLTxxxCxxLxxaLxxxx (SEQ ID NO: 6),        wherein “x” denotes any amino acid, and “a” denotes an aliphatic        amino acid (A-type LRR).

Particularly preferred is furthermore a collection, wherein at least oneof said modules comprises the LRR consensus sequence

-   -   xLxELxLxxNxLGDxGaxxLxxxLxxPxx (SEQ ID NO: 7),        wherein “x” denotes any amino acid, and “a” denotes an aliphatic        amino acid (B-type LRR).

Most preferred is a collection, wherein one or more of the positionsdenoted “x” and/or “±” are randomised.

Further preferred is a collection, wherein the cysteine residue atposition 10 in the A-type LRR consensus sequence is replaced by ahydrophilic amino acid residue, and wherein the cystein residue atposition 17 is replaced by a hydrophobic amino acid residue.

A hydrophilic an acid residue may be taken from the list of Ser, Thr,Tyr, Gln, and Asn.

A hydrophobic amino acid residue may be taken from the list of Ala, Ile,Leu, Met, Phe, Trp, and Val.

Compared to single-chain Fv or conventional antibodies, severaladvantages can be enumerated. Whereas disulfide bridges are crucial forthe stability of most antibodies (Proba et al., 1997), no disulfidebonds are required in LRR proteins, which makes intracellularapplications possible.

Therefore, new binding molecules can be generated for application in areducing environment. This could become an enormously powerful tool inelucidating the function of the numerous proteins identified by thegenome sequencing projects by direct inhibition in the cytosol. As formany applications in biotechnology large amounts of expressed andcorrectly folding proteins are required, a production in E. coli ispreferable but very difficult for antibodies which evolved in theoxidising extracellular environment. In contrast, folding or refoldingof RI variants are more efficient as they are naturally found in thecytosol (see Example 1).

In a further preferred embodiment of a collection according to thepresent invention, one or more of the amino acid residues in an ankyrinor LRR repeat module as described above are exchanged by an amino acidresidue found at the corresponding position in a corresponding naturallyoccurring repeat unit. Preferably, up to 30% of the amino acid residuesare exchanged, more preferably, up to 20%, and most preferably, up to10% of the amino acid residues are exchanged.

Particularly preferred is a collection, wherein said set consists of onetype of repeat modules.

The term “type of repeat module” refers to the characteristics of amodule determined by the length of the module, the number andcomposition of its “fixed positions” as well as of its “randomisedpositions”. “Different types of modules” may differ in one or more ofsaid characteristics.

Further preferred is a collection, wherein said set consists of twodifferent types of repeat modules.

In a still further preferred embodiment, the present invention relatesto a collection, wherein said set comprises two different types ofconsecutive repeat modules as pairs in said repeat proteins.

Most preferred is a collection, wherein said two different types ofmodules are based on said A-type LRR and B-type LRR.

Further preferred is a collection, wherein the amino acid sequences ofthe repeat modules comprised in said set are identical for each saidtype except for the randomised residues.

Yet further preferred is a collection, wherein the nucleic acidsequences encoding the copies of each said type are identical except forthe codons encoding amino acid residues at positions being randomised.

Particularly preferred is a collection, wherein the nucleic acidmolecules encoding said repeat proteins comprise identical nucleic acidsequences of at least 9 nucleotides between said repeat modules.

Said “identical nucleic acid sequences of at least 9 nucleotides” may bepart of the end of only one repeat module, or be formed by the ends oftwo adjacent repeat modules, or may be part of a (poly)peptide linkerconnecting two repeat modules.

In a further preferred collection according to the present invention,the nucleic acid molecules encoding said repeat proteins compriseidentical nucleic acid sequences of at least 9 nucleotides between saidpairs.

Said “identical nucleic acid sequences of at least 9 nucleotides” may bepart of the end of only one pair of repeat modules, or be formed by theends of two adjacent pairs of repeat modules, or may be part of a(poly)peptide linker connecting two pairs of repeat modules.

Most preferable is a collection, wherein each of the nucleic acidsequences between said modules, or said pairs, comprises a restrictionenzyme recognition sequence.

The term “restriction enzyme recognition sequence” refers to a nucleicacid sequence being recognised and cleaved by a restrictionendonuclease. Said restriction enzyme recognition sequence may bedivided symmetrically between the 3′ and 5′ ends (e.g. 3 nucleotides ofa 6 base pair recognition sequence on both ends), or non-symmetrically(e.g. 2 nucleotides on one end, 4 on the corresponding end).

Particularly preferred is a collection, wherein each of the nucleic acidsequences between said modules, or said pairs, comprises a nucleic acidsequence formed from cohesive ends created by two compatible restrictionenzymes.

The term “compatible restriction enzymes” refers to restriction enzymeshaving different recognition sequences but forming compatible cohesiveends when cleaving double stranded DNA. After re-ligation of sticky-enddouble-stranded DNA fragments produced from two compatible restrictionenzymes, the product DNA does no longer exhibit the recognitionsequences of both restriction enzymes.

In a further most preferred embodiment of the collection of the presentinvention, said identical nucleic acid sequences allow a PCR-basedassembly of the nucleic acid molecules encoding said repeat proteins.

In a most preferred embodiment of the collection according to thepresent invention, said repeat proteins comprise one or more pairs ofmodules based on said A-type LRR and B-type LRR, wherein each of saidpairs has the sequenceRLE1L112DLTEAG4KDLASVLRSNPSLREL3LS3NKLGDAGVRLLLQGLLDPGT (SEQ ID NO: 8),

wherein 1 represents an amino acid residue selected from the group:

D, E, N, Q, S, R, K, W and Y;

wherein 2 represents an amino acid residue selected from the group:

N, S and T;

wherein 3 represents an amino acid residue selected from the group:

G, S, D, N, H and T; and

wherein 4 represents an amino acid residue selected from the group:

L, V and M.

Most preferably, each of said pairs of modules is encoded by the nucleicacid molecule

-   -   CGC CTG GAG 111 CTG 111 CTG 111 111 222 GAC CTC ACC GAG GCC GGC        444 AAG GAC CTG GCC AGC GTG CTC CGC TCC AAC CCG AGC CTG CGG GAG        CTG 333 CTG AGC 333 AAC AAG CTC GGC GAT GCA GGC GTG CGG CTG CTC        TTG CAG GGG CTG CTG GAC CCC GGC AGG (SEQ ID NO: 9)        wherein 111 represents a codon encoding an amino acid residue        selected from the group:        D, E, N, Q, S, R, K, W and Y;        wherein 222 represents a codon encoding an amino acid residue        selected from the group:        N, S and T;        wherein 333 represents a codon encoding an amino acid residue        selected from the group:        G, S, D, N, H and T; and        wherein 444 represents a codon encoding an amino acid residue        selected from the group:        L, V and M.

In another preferred embodiment one or more of the amino acid residuesin at least one pair of modules as listed above are exchanged by anamino acid residue found at the corresponding position in a naturallyoccurring LRR.

In yet another preferred embodiment, one or more of the amino acidcodons in at least one pair of modules as listed above are exchanged bya codon encoding an amino acid residue found at the correspondingposition in a naturally occurring LRR.

Preferably, up to 30% of the amino acid residues, or amino acid codons,respectively, are exchanged, more preferably, up to 20%, and mostpreferably, up to 10% are exchanged.

In yet another preferred embodiment, one or more of the amino acidcodons in at least one pair of modules as listed above are exchanged bya codon encoding an amino acid residue found at the correspondingposition in a naturally occurring LRR.

In a further preferred embodiment, the present invention relates to acollection of recombinant nucleic acid molecules comprising a collectionof nucleic acid molecules according to the present invention.

In the context of the present invention, the term “recombinant nucleicacid molecule” refers to a RNA or DNA molecule which comprises a nucleicacid sequence encoding said repeat protein and further nucleic acidsequences, e.g. non-coding sequences.

In a still further preferred embodiment, the present invention relatesto a collection of vectors comprising a collection of nucleic acidmolecules according to the present invention, or a collection ofrecombinant nucleic acid molecules according to the present invention.

A vector according to the present invention may be a plasmid, phagemid,cosmid, or a virus- or bacteriophage-based vector, and may be a cloningor sequencing vector, or preferably an expression vector, whichcomprises all elements required for the expression of nucleic acidmolecules from said vector, either in prokaryotic or eukaryoticexpression systems. Vectors for cloning, sequencing and expressingnucleic acid molecules are well known to any one of ordinary skill inthe art. The vectors containing the nucleic acid molecules of theinvention can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilised for prokaryotic cells,whereas, e.g., calcium phosphate or DEAE-Dextran mediated transfectionor electroporation may be used for other cellular hosts; see Sambrook etal. (1989).

Such vectors may comprise further genes such as marker genes which allowfor the selection of said vector in a suitable host cell and undersuitable conditions. Preferably, the nucleic acid molecules of theinvention are operatively linked to expression control sequencesallowing expression in prokaryotic or eukaryotic cells. Expression ofsaid nucleic acid molecules comprises transcription of thepolynucleotide into a translatable mRNA. Regulatory elements ensuringexpression in eukaryotic cells, preferably mammalian cells, are wellknown to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and, optionally, a poly-Asignal ensuring termination of transcription and stabilization of thetranscript, and/or an intron further enhancing expression of saidnucleic acid molecule. Additional regulatory elements may includetranscriptional as well as translational enhancers, and/ornaturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in prokaryotic host cellscomprise, e.g., the pL, lac, trp or tac promoter in E. coli, andexamples for regulatory elements permitting expression in eukaryotichost cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-,RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or aglobin intron in mammalian and other animal cells. Beside elements whichare responsible for the initiation of transcription, such regulatoryelements may also comprise transcription termination signals, such asthe SV40-poly-A site or the tk-poly-A site, downstream of the nucleicacid molecule. Furthermore, depending on the expression system usedleader sequences capable of directing the (poly)peptide to a cellularcompartment or secreting it into the medium may be added to the codingsequence of the nucleic acid molecule of the invention and are wellknown in the art. The leader sequence(s) is (are) assembled inappropriate phase with translation, initiation and terminationsequences, and preferably, a leader sequence capable of directingsecretion of translated protein, or a portion thereof, into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusion protein including a C- or N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. In this context, suitable expression vectors are known in theart such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia),pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL) orpCI (Promega) or more preferably pTFT74 (Ge et al., 1995) or a member ofthe pQE series (Qiagen). Furthermore, the present invention relates tovectors, particularly plasmids, cosmids, viruses and bacteriophages usedconventionally in genetic engineering that comprise the polynucleotideof the invention. Preferably, said vector is an expression vector.Methods which are well known to those skilled in the art can be used toconstruct recombinant viral vectors; see, for example, the techniquesdescribed in Sambrook et al., Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel et al., CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y. (1989).

Furthermore, the invention relates to a collection of host cellscomprising a collection of nucleic acid molecules according to thepresent invention, a collection of recombinant nucleic acid moleculesaccording to the present Invention, or a collection of vectors accordingto the present invention.

In the context of the present invention the term “host cell” may be anyof a number commonly used in the production of heterologous proteins,including but not limited to bacteria, such as Escherichia coli (Ge etal., 1995), or Bacillus subtilis (Wu et al., 1993a), fungi, such asyeasts (Horwitz et al., 1988; Ridder et al., 1995) or filamentous fungus(Nyyssönen et al., 1993), plant cells (Hiatt, 1990; Hiatt and Ma, 1993;Whitelam et al., 1994), insect cells (Potter et al., 1993; Ward et al.,1995), or mammalian cells (Trill et al., 1995).

In another embodiment, the present invention relates to a collection ofrepeat proteins encoded by a collection of nucleic acid moleculesaccording to the present invention, by a collection of vectors accordingto the present invention, or produced by a collection of host cellsaccording to the present invention.

Furthermore, the present invention relates to a method for theconstruction of a collection of nucleic acid molecules according to thepresent invention, comprising the steps of

-   -   (a) identifying a repeat unit from a repeat protein family;    -   (b) identifying framework residues and target interaction        residues in said repeat unit;    -   (c) deducing at least one type of repeat module comprising        framework residues and randomised target interaction residues        from at least one member of said repeat protein family; and    -   (d) constructing nucleic acid molecules each encoding a repeat        protein comprising two or more copies of said at least one type        of repeat module deduced in step (c).

The modes how this method is to be carried out are explained above inconnection with the embodiment of the collection of nucleic acidmolecules of the present invention. Descriptions of two such modes areillustrated in the example.

In a preferred embodiment of this method, said at least one repeatmodule deduced in step (c) has an amino acid sequence, wherein at least70% of the amino acid residues correspond either

-   (i) to consensus amino acid residues deduced from the amino acid    residues found at the corresponding positions of at least two    naturally occurring repeat units; or-   (ii) to the amino acid residues found at the corresponding positions    in a naturally occurring repeat unit.

Further preferred is a method for the production of a collection of(poly) peptides/proteins according to the present invention, comprisingthe steps of

-   (a) providing a collection of host cells according to the present    invention; and-   (b) expressing the collection of nucleic acid molecules comprised in    said host cells.

Particularly preferred is a method for obtaining a repeat protein havinga predetermined property, comprising the steps of

-   (a) providing a collection of repeat proteins according to the    present invention; and-   (b) screening said collection and/or selecting from said collection    to obtain at least one repeat protein having said predetermined    property.

The diverse collection of repeat proteins may be provided by severalmethods in accordance with the screening and/or selection system beingused, and may comprise the use of methods such as display on the surfaceof bacteriophages (WO 90/02809; Smith, 1985; Kay et al., 1996; Dunn,1996) or bacterial cells (WO 93/10214), ribosomal display (WO 91/05058;WO 98/48008; Hanes et al., 1998), display on plasmids (WO 93/08278) orby using covalent RNA-repeat protein hybrid constructs (WO 00/32823),intracellular expression and selection/screening such as by proteincomplementation assay (WO 98/341120; Pelletier et al., 1998). In allthese methods, the repeat proteins are provided by expression of acorresponding collection of nucleic acid molecules and subsequentscreening of the repeat proteins followed by identification of one ormore repeat proteins having the desired property via the geneticinformation connected to the repeat proteins.

In the context of the present invention the term “predeterminedproperty” refers to a property, which one of the repeat proteins out ofthe collection of repeat proteins should have, and which forms the basisfor screening and/or selecting the collection. Such properties compriseproperties such as binding to a target, blocking of a target, activationof a target-mediated reaction, enzymatic activity, and furtherproperties, which are known to one of ordinary skill. Depending on thetype of desired property, one of ordinary skill will be able to identifyformat and necessary steps for performing screening and/or selection.

Most preferably, the present invention relates to a method, wherein saidpredetermined property is binding to a target.

In another embodiment, the invention relates to a repeat protein from acollection according to the present invention.

Preferably said repeat protein has been obtained by the above-describedmethod and has one of the predetermined properties.

Furthermore, the present invention relates to a nucleic acid moleculeencoding the repeat protein according to the present invention.

In yet another embodiment, the present invention relates to a vectorcontaining the nucleic acid molecule according to the present invention.

The present invention relates also to pharmaceutical compositionscomprising a repeat protein from a collection of the present inventionor a nucleic acid molecule encoding said repeat protein, and optionallya pharmaceutically acceptable carrier and/or diluent.

Examples of suitable pharmaceutical carriers are well known in the artand include phosphate buffered saline solutions, water, emulsions, suchas oil/water emulsions, various types of wetting agents, sterilesolutions etc. Compositions comprising such carriers can be formulatedby well known conventional methods. These pharmaceutical compositionscan be administered to the subject at a suitable dose. Administration ofthe suitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical,intradermal, intranasal or intrabronchial administration. The dosageregimen will be determined by the attending physician and clinicalfactors. As is well known in the medical arts, dosages for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the regimen as a regularadministration of the pharmaceutical composition should be in the rangeof 1 μg to 10 mg units per day. If the regimen is a continuous infusion,it should also be in the range of 1 μg to 10 mg units per kilogram ofbody weight per minute, respectively. Progress can be monitored byperiodic assessment. Dosages will vary but a preferred dosage forintravenous administration of DNA is from approximately 10⁶ to 10¹²copies of the DNA molecule. The compositions of the invention may beadministered locally or systemically. Administration will generally beparenterally, intravenously; DNA may also be administered directly tothe target site, e.g., by biolistic delivery to an internal or externaltarget site or by catheter to a site in an artery. Preparations forparenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.Furthermore, the pharmaceutical composition of the invention maycomprise further agents such as interleukins or interferons depending onthe intended use of the pharmaceutical composition.

The repeat proteins comprised in the pharmaceutical compositions of thepresent invention can comprise a further domain, said domain beinglinked by covalent or non-covalent bonds. The linkage can be based ongenetic fusion according to the methods known in the art and describedabove or can be performed by, e.g., chemical cross-linking as describedin, e.g., WO 94/04686. The additional domain present in the fusionprotein comprising the peptide, polypeptide or antibody employed inaccordance with the invention may preferably be linked by a flexiblelinker, advantageously a polypeptide linker, wherein said polypeptidelinker comprises plural, hydrophilic, peptide-bonded amino acids of alength sufficient to span the distance between the C-terminal end ofsaid further domain and the N-terminal end of the repeat protein or viceversa. The above described fusion protein may further comprise acleavable linker or cleavage site for proteinases.

Furthermore, said further domain may be of a predefined specificity orfunction. In this context, it is understood that the repeat proteinspresent in the pharmaceutical composition according to the invention maybe further modified by conventional methods known in the art. Thisallows for the construction of fusion proteins comprising the repeatprotein of the invention and other functional amino acid sequences,e.g., nuclear localization signals, transactivating domains, DNA-bindingdomains, hormone-binding domains, protein tags (GST, GFP, h-myc peptide,FLAG, HA peptide) which may be derived from heterologous proteins. Thus,administration of the composition of the invention can utilize unlabeledas well as labeled (poly)peptides or antibodies.

Further preferred is a nucleic acid molecule encoding a pair of repeatmodules for the construction of a collection according to the presentinvention, wherein said nucleic acid molecule is:

-   -   CGC CTG GAG 111 CTG 111 CTG 111 111 222 GAC CTC ACC GAG GCC GGC        444 AAG GAC CTG GCC AGC GTG CTC CGC TCC AAC CCG AGC CTG CGG GAG        CTG 333 CTG AGC 333 AAC AAG CTC GGC GAT GCA GGC GTG CGG CTG CTC        TTG CAG GGG CTG GTG GAC CCC GGC ACG (SEQ ID NO: 10),        wherein 111 represents a codon encoding an amino acid residue        selected from the group:        D, E, N, S, R, K, W and Y;        wherein 222 represents a codon encoding an amino acid residue        selected from the group:        N, S and T;        wherein 333 represents a codon encoding an amino acid residue        selected from the group:        G, S, D, N, H and T; and        wherein 444 represents a codon encoding an amino acid residue        selected from the group:        L, V and M.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries, using for example electronic devices. For example database“PubMed” (Sequeira et al., 2001) may be utilised which is available onthe Internet.

An overview of patent information in biotechnology and a survey orrelevant sources of patent information useful for retrospectivesearching and for current awareness is given in Berks, (1994).

FIGURES

FIG. 1. Schematic representation of the terms “Repeat Protein”, “RepeatDomains”, “Non-repeat Domain”, “Repeat Module”, “Capping Modules”, and“Linker”.

FIG. 2a . Examples of leucine-rich repeat proteins featuring only arepeat domain (1A4Y) or both a repeat domain and a non-repeat domain(1D0B).

FIG. 2b . Examples of ankyrin repeat proteins featuring only a repeatdomain (1AWC) or both a repeat domain and a non-repeat domain (1DCQ).

FIG. 2c . Crystal Structure of the Pig Liver Ribonuclease Inhibitor(Kobe and Deisenhofer, 1993).

FIG. 2d . Crystal Structure of the Yeast ma1p GTPase-activating Protein(Hillig at al., 1999)

FIG. 2e . Crystal Structure of the Listens InIB Protein (Marino et al.,1999).

FIG. 2f . Crystal Structure of the Human Spliceosomal Protein U2A′(Price et al., 1998).

FIG. 2g . Crystal Structure of the Human Transcription Factor InhibitorIκBα (Huxford et al., 1998).

FIG. 2h . X-ray structure of the ankyrin repeat domain of the mouseGA-binding protein beta 1 subunit [pdb entry 1AWC (Batchelor et al.,1998)]. The N- and C-termini of the domain are labeled. This image hasbeen created using MOLMOL (Koradi et al., 1996),

FIG. 3. Examples of naturally occurring repeat units and capping units.A leucine-rich repeat protein (1A4Y) and an ankyrin repeat protein(1AWC) are shown.

FIG. 4a . β/α-Fold of the LRR unit from Pig Ribonuclease Inhibitor(Residue 423 to 450),

FIG. 4b . Leucines and Positions of Amino Acids Emanating from theβ-strand of a LRR unit from Pig Ribonuclease Inhibitor (Residue 86 to112).

FIG. 4c . Structural description of an ankyrin repeat unit. A: Sideview.B: Topview Interacting residues are depicted as “balls and sticks”.These pictures were made using the third repeat of the GA-bindingprotein (pdb entry 1/AWC; Batchelor et al., 1998) displayed with MOLMOL(Koradi al., 1996).

FIG. 4d . A subset of the framework residues of a LRR unit is shown as“ball and sticks”. The numbering refers to the positions within a LRRunit.

FIG. 4e . A subset of the target interaction residues of a LRR unit isshown as “ball and sticks”. The numbering refers to the positions withina LRR unit.

FIG. 4f . A model of a LRR repeat module pair is shown. The numberingrefers to the positions within the derived LRR repeat motif pair.

FIG. 5e . Internal Amino Acid Alignment of Human Placental RibonucleaseInhibitor (SEQ ID NO: 53).

FIG. 5b . Consensus (SEQ ID NO: 8) Defined on the Basis of allRibonuclease Inhibitor Sequences.

FIG. 5c . Statistical analysis of the most frequent amino acids at onepositions in the A-type repeat units of mammalian R1. Each of the fourboxes represents one of SEQ ID NOS 54, 55, 56 and 57 (respectively, inorder of appearance from top to bottom) The sequences of the A-typerepeat units of individual RIs are each presented in a box of 7 time 28amino acids, since a preliminary analysis indicates that each RIpossesses seven A-type repeat units having each a length of 28 aminoacids. The consensus sequence based on the aligned repeat units is undereach box. The consensus sequence for SEQ ID NO: 54 is SEQ ID NO: 78; forSEQ ID NO: 55 is SEQ ID NO: 79; for SEQ ID NO: 56 is SEQ ID NO: 80; and,for SEQ ID NO: 57 is SEQ ID NO:81.

FIG. 5d Statistical analysis of the most frequent amino acids at onepositions in the B-type repeat units of mammalian RI. Each of the fourboxes represents one of SEQ ID NOS 58, 59, 60 and 61 (respectively, inorder of appearance from top to bottom). The sequences of B-type repeatunits of individual RIs are each presented in a box of 7 times 29 aminoacids, since a preliminary analysis indicates that each RI possessesseven B-type repeat units having each a length of 29 amino acids. Theconsensus sequence based on the aligned repeat units is under each box.The consensus sequence for SEQ ID NO: 58 is SEQ ID NO: 82; for SEQ IDNO: 59 is SEQ ID NO: 83; for SEQ ID NO: 60 is SEQ ID NO: 84; and, forSEQ ID NO: 61 is SEQ ID NO: 85.

FIG. 6. Restriction Enzyme Recognition Sites and Encoded Amino Acids.The DNA recognized by BssHII codes for alanine and arginine (A and R) inthe first reading frame. Accordingly. MluI codes for threonine andarginine (T and R) in the first reading frame. Combination of DNAmolecules cut with BssHII and MluI give a new combined site notrecognized by either restriction enzyme and coding for alanine andarginine (A and R).

FIGS. 7a to 7c . Cloning of the library of repeat modules.

FIG. 8. DNA sequence (SEQ ID NO: 62) and translated amino acids (SEQ IDNO: 63) of the NcoI-HindIII insert in plasmid pTFT_N1CL. Theabbreviation pTFT refers to all plasmids derived from pTFT74 (Ge et al.,1995). The abbreviation N1CL refers to an insert containing anN-terminal module, 1 repeat module, a C-terminal module, and a linkersequence.

FIGS. 9a to 9c . Diagrams of plasmids pTFT_N, pQE_N1C, and pQE-pD_N2C.The nomenclature is as described in the caption of FIG. 8. The name ofplasmids derived from pQE30 (Qiagen) always starts with pQE. Theabbreviation pD refers to lambda phage protein D (Forrer and Jaussi,1998).

FIGS. 10a and 10b . DNA sequence (SEQ ID NO: 64 and encoded protein (SEQID NO:65) of the NcoI-HindIII insert of plasmid pQE_N4C clone D17.

FIG. 11a . High-level expression of randomly chosen members of thepD_N2C library (A2, A10, . . . ). XL1-Blue cells containing one of thelibrary expression plasmid pQE-pD_N2C were grown at 37° C. to an OD₆₀₀=1and induced for 1 h with 1 mM IPTG. The collected cells were resuspendedin TBS₅₀₀, sonicated, and centrifuged. Samples corresponding to thesupernatant (S) or pellet (P) of 40 microliters of cell culture wereseparated on a 15% SDS-PAGE and stained with Coomassie Blue. The clonesare designated A2, A10, and so on. Ap1 and Ap2 are pools of 10individual clones; Y: truncated pD_N2C (26 kDa), X: pD_N2C (33 kDa).

FIG. 11b . High-level expression of randomly chosen members of the N2C(C1, C2, . . . ) and pD_N4C (B9, B21) libraries as described in FIG. 11a; *: N2C (22 kDa), #: pD_N4C (45 kDa).

FIG. 11c . High-level expression of randomly chosen members of the N4C(D11, D15, . . . ) library as described in FIG. 11a ; Z: N4C (34 kDa).

FIG. 11d , High-level expression of randomly chosen members of the N4C(D11, D15, . . . ) library as described in FIG. 11a but growth at 25°C.; Z: N4C (34 kDa).

FIG. 12a , Western blot analysis of high-level expression of members ofthe pD_N2C library (A2, A10, A15) after expression at either 25° C. or30° C. Protein was prepared as for FIG. 11a , Antibody anti-RGS-His wasused in 1:5000 dilution following the manufacturer's protocol (Qiagen);Y: truncated pD_N2C (26 kDa), X: pD_N2C (33 kDa).

FIG. 12b . Western blot analysis of high-level expression of randomlychosen members of the pD_N4C library (B9, B21, BP which is a pool) afterexpression at either 37° C. or 25° C.; #: pD_N4C (45 kDa).

FIG. 12c . Western blot analysis of high-level expression of somemembers of the N2C library (C1, C3, C7) after expression at either 37°C. or 25° C. Protein was prepared as for FIG. 11a , Antibody anti-FlagM2 was used in 1:1000 dilution following the manufacturer's protocol(Sigma); *: N2C (22 kDa).

FIG. 12d . Western blot analysis of high-level expression of somemembers of the N2C library (D17, D19, D22) after expression at either37° C. or 25° C.; Z: N4C (34 kDa).

FIG. 13. His-tag purification under native conditions of a randomlychosen leucine-rich repeat protein according to the present invention.Lane M shows the molecular size marker (in kDa), lane FT shows theunbound fraction, and lanes 0 to 6 show different elution fractions. Thearrow indicates the position of the expected protein.

FIG. 14. His-tag purification under denaturing conditions includingrefolding of the repeat proteins in the purification column. Lanes 1 to6 show the unbound fractions of six leucine-rich repeat proteinsaccording to the present invention. Lanes 7 to 12 show the peak elutionfractions of the same six proteins. The arrow indicates the position ofthe expected proteins.

FIG. 15. Circular dichroism spectrometry of a randomly chosenleucine-rich repeat protein according to the present invention.

FIG. 16. Size exclusion chromatography of a randomly chosen leucine-richrepeat protein according to the present invention. The sample wasanalysed on a Superose 12 column.

FIG. 17. DNA recognition sequences of the restriction enzymes used forthe cloning of ankyrin repeat proteins according to the presentinvention. Type II restriction enzymes cleave DNA within a palindromicrecognition site, while type IIs restriction enzymes cut outside anon-palindromic recognition site. Two type IIs restriction enzymes (BpiI(SEQ ID NO: 66) and BsaI (SEQ ID NO: 67)) were used to ligate ankyrinrepeat modules with each other in a directed manner by virtue of theircompatible overhangs (see FIG. 18, Table 2 and Table 3), generatingseamless connections of a ankyrin repeat module to the next one. Thesetype IIs restriction enzymes were also used to link the N- and theC-terminal ankyrin capping modules with the ankyrin repeat modulesseparating them. BamHI and HindIII were used for the cloning of theankyrin repeat proteins constructed according to the present invention(containing a N-terminal ankyrin capping module, two or more ankyrinrepeat modules and a C-terminal ankyrin capping module) into plasmidpQE30 (QIAgen, Germany). The pattern of restriction is indicated foreach enzyme by solid lines.

FIG. 18. Schematic view of the stepwise elongation of the N-terminalankyrin capping module with ankyrin repeat modules on DNA level. TheN-terminal ankyrin capping module is elongated by ankyrin repeat modulesto the required length, followed and ended by the addition of theC-terminal ankyrin capping module.

FIG. 19. Consensus “A” (SEQ ID NO: 68) (obtained after SMART analysis),the consensus used for the BLAST search (SEQ ID NO: 70) (circularlypermutated consensus “A” where missing residues have been taken from aconsensus of ankyrin repeat units of ankyrin repeat proteins with knownthree dimensional structure), consensus “B” (SEQ ID NO: 69) (derivedafter BLAST search) as well as consensus “C” (SEQ ID NO: 71) (finallyobtained considering various parameters mentioned in EXAMPLE 2) arelisted to illustrate the stepwise definition of the ankyrin repeat unitconsensus. For consensus “A” and “B”, residues reaching 20% frequency ata given position are displayed. In consensus “C”, several amino acidsare displayed at positions to which the latter amino acids fittedequally well.

FIG. 20. The sequence of the ankyrin repeat motif (SEQ ID NO: 72) (i.e.the basis of all ankyrin repeat modules of EXAMPLE 2) and the respectiveposition numbers of the amino acids are displayed. In addition, theexpected secondary structures (α meaning α-helix, β meaning β-sheet) areindicated. The six positions denoted “x” were defined as targetinteraction residues which were allowed to be any of the amino acids A,D, E, F, H, I, K, L, M, N, O, R, S, T, V, W and Y. The remainingpositions were defined to be framework residues defined by consensus “C”(cf. FIG. 19). At position 26, any out of the three amino acidshistidine, tyrosine or asparagine were allowed. For cloning reasons theankyrin repeat motif is based on a circularly permutated consensus “C”(cf. FIG. 19). To match the consensus numbering scheme used in FIG. 19and used by Sedgwick and Smerdon (1999), the numbers were circularlypermutated in parallel with the consensus sequence.

FIG. 21, Alignment of the randomly chosen clone “E3-5” (SEQ ID NO: 73)constructed according to the present invention. The amino acid sequenceof E3-5, a protein having 3 ankyrin repeat modules (FIG. 20) between theN- and the C-terminal ankyrin capping modules, is aligned to mouseGA-binding protein beta 1 (SEQ ID NO: 74). The latter is the proteinshowing highest homology to E3-5 among known ankyrin repeat proteins.The sequences were aligned using the command “gap” of GCG (Womble, D.D., 2000) with default values and the sequence comparison matrixBlosum62. Over all, the two molecules showed 67% residue identity and71% residue homology. Positions corresponding to randomised positions inthe repeat motif (cf. FIG. 20) are marked with an asterisk above. TheN-terminal and C-terminal ankyrin capping modules are overlined, thethree ankyrin repeat modules underlined.

FIG. 22. High-level expression of differently sized ankyrin repeatproteins generated according to the present invention [BamHI/HindIIIcloned into plasmid pQE30 (QIAgen); expressed in E. coli XL1-Blue(Stratagene)]. Of each library of N2C, N3C and N4C, two randomly chosenclones were tested. The abbreviation N2C refers to an N-terminal ankyrincapping module, two ankyrin repeat modules and a C-terminal ankyrincapping module being connected using the cloning strategy stated in FIG.17 and FIG. 18. N3C and N4C are named accordingly to their content ofthree or four ankyrin repeat modules between their N- and C-terminalankyrin capping modules. Expression was performed as described inEXAMPLE 2. Samples corresponding to 30 μl of culture were taken atvarious timepoints and separated on 16% SDS-PAGE (Coomassie stained).Lane 1: Molecular marker (size indicated in kDa); Lane 2-7: two N2C, twoN3C and two N4C clones just before induction; Lane 8-13: same as lane2-7 but after 2.5 hours induction: Lane 14-19: same as lane 2-7 butafter 4 hours induction.

FIG. 23. His-tag purification of a randomly chosen ankyrin repeatprotein generated according to the present invention. A 15% SOS-PAGEshowing different fractions of the purification procedure is depicted.E3-5, an N3C clone, was expressed and purified as described in EXAMPLE2. Lane 1 represents 0.6 μl of the collected cell lysate flow throughwhich was not bound by the Ni-NTA columns. Lane 2 represents 0.6 μl offirst 800 μl column washing fraction. Lane 3 represent 0.6 μl of thelast 800 μl washing fraction. Lanes 4, 5, 6, 7, 8 and 9 represent 0.6 μlof the subsequent elution steps (800 μl each) of the ankyrin repeatprotein. Lane 10 shows the molecular marker (sizes in kDa).

FIG. 24. Size exclusion chromatography of a randomly chosen ankyrinrepeat protein generated according to the present invention (E3-5, a N3Cmolecule; cf. FIG. 22). The sample was analysed on a Superdex 75 column(Amersham Pharmacia Biotech, USA) using a Pharmacia SMART system at aflow rate of 60 μl/min and TBS 150 (50 mM Tris-HCl, pH 7.5; 150 mM NaCl)as running buffer. Standards were Beta-amylase and the phage proteinsSHP of phage 21 and pD of phage Lambda. The apparent masses of thestandards are indicated in the figure. The apparent mass of 200 kDa forBeta-amylase is not indicated, as the protein eluted in the void volume.

FIG. 25. Circular dichroism spectra of a randomly chosen ankyrin repeatprotein generated according to the present invention (E3-5, a N3Cmolecule). The spectra were recorded either in 10 mM sodium phosphatebuffer pH 6.5 (native) or 20 mM sodium phosphate buffer pH 6.5 and 8MGuanidinium hydrochloride (denatured) using a Jasco J-715 instrument[Jasco, Japan; 10 nm/s, 8 sec response, 0.2 nm data pitch, 2 nm bandwidth, 195-250 nm (native) or 212-250 nm (denatured), threeaccumulations, measurements in triplicates, 1 mm cuvette]. The CD signalwas converted to mean residue elipticity using the concentration of thesample determined spectrophotometrically at 280 nm under denaturingconditions. E3-5 shows an alpha-helical spectrum under native conditionswith minima at 208 nm and 222 nm. The secondary structure is lost in 6 MGuanidinium hydrochloride.

FIG. 26. Denaturation behaviour of randomly chosen ankyrin repeatproteins generated according to the present invention (cf. FIG. 22). TheCD values at 220 nm are shown over guanidinium hydrochlorideconcentration for the different proteins. The different proteins wereincubated with different concentrations of guanidinium hydrochloride in20 mM NaPO4 pH6.5, 100 mM NaCl, overnight at room temperature. Thecircular dichroism signal at 220 nm was measured for each sample intriplicates (conditions as indicated in EXAMPLE 2). The secondarystructure is lost only at high concentrations of denaturing agentindicating a high stability of the tested proteins.

FIG. 27. Crystals of a randomly chosen ankyrin repeat protein generatedaccording to the present invention (E3-5, a N3C library member of FIG.22). The crystal was grown in five days at 20° C. in 20% PEG 6000, 100mM MES/NaOH pH 6.0, hanging droplet (2 μl protein and 2 μl buffer mixed;500 μl buffer reservoir) from a solution of 9 mg Protein per ml in TBS50 (60 mM TrisHCl, pH 8.0, 50 mM NaCl).

The examples illustrate the invention.

EXAMPLES

Unless stated otherwise in the examples, all recombinant DNA techniquesare performed according to described protocols (Sambrook et al., 1989 orAusubel et al., 1994), Databases used were

Genbank

-   -   National Center for Biotechnology Information, National Library        of Medicine, Bethesda, USA        Swiss-Prot    -   Swiss Institute of Bioinformatics, Geneva, Switzerland        Protein Data Base    -   Center for Molecular Biophysics and Biophysical Chemistry at        Rutgers, N.J., USA        Simple Modular Architecture Research Tool (SMART)    -   EMBL, Heidelberg, Germany        1. Collection of Repeat Proteins Comprising Repeat Modules        Derived from Repeat Units of Mammalian Ribonuclease Inhibitors

This example describes the construction of a collection of leucine-richrepeat proteins derived from mammalian ribonuclease inhibitors (RI).This scaffold was chosen, since extraordinarily tight interactions inthe femtomolar range have been reported for the binding of angiogenin byRI (Lee et al., 1989) and RNase A by RI (Kobe and Deisenhofer, 1996).

As the RI amino acid sequence showed a characteristic pattern of twoalternating, different but homologous repeat units, termed A- and B-typeLRR repeat unit (Kobe and Deisenhofer, 1994), two according repeatmotifs were derived and used to build a repeat domain. The assembly of aLRR repeat motif of type A with a LRR repeat motif of type B ishenceforth referred to as RI repeat motif pair. A model of a repeatmodule pair comprising a RI repeat motif pair is shown (FIG. 4f ). Thisexample demonstrates the use of more than one repeat motif to build arepeat domain, which is in contrast to example 2 where only one repeatmotif is used.

1) Deriving Preliminary Repeat Sequence Motifs of Mammalian RI

The protein sequences of human RI (accession number P13489. Lee et al.,1988) and pig RI (P10775, Hofsteenge et al., 1988) were used to searchfor homologous sequences. The complete protein sequence of the rat RI(P29315, Kawanomoto et al., 1992) and mouse RI protein were found(AAK68859, unpublished).

The repeat units of the obtained RI protein sequences were aligned using“FastA” implemented in the GCG® Wisconsin Package™ (Accelrys, USA). Theprotein sequence of human RI is shown (FIG. 5a ) and the LRR patterncharacterised by leucines or other aliphatic residues at positions 2, 5,7, 12, 20, and 24 (Kobe and Deisenhofer, 1994) is highlighted. The mostabundant amino acid for each position was calculated for the human,mouse, pig, and rat RI sequences (FIGS. 5c and 5d ). A first RI repeatmotif pair was defined by amino acids occurring in 50% (cf. FIG. 5c and5d ) or more of the cases at a given position

A-type LRR consensus (SEQ ID NO: 11)1  3  5  7  9  11 13 15 17 19 21 23 25 27 -LE-L-L--C-LT-A-C--L-SVL----B-type LRR consensus (SEQ ID NO: 12)1  3  5  7  9  11 13 15 17 19 21 23 25 27 29SL-EL-LS-N-LGD-G---LC-GL--P-C

For a threshold of 40% or more identical amino acids at a given positionthe RI repeat motif pair was defined by the following amino acidsequence

A-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27+LE-L-L--C-LTAA-C-DL-SVLRAN- where + is R or K (SEQ ID NO: 13)B-type LRR consensus 1..3  5  7  9  11 13 15 17 19 21 23 25 27 29(SEQ ID NO: 14)

Similarly, for a threshold of 30% or more identical amino acids at agiven position the RI repeat motif pair was defined by the followingamino acid sequence

A-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27+LE-LWL-DCGLTAAGCKDLCSVLRAN- where + is R or K (SEQ ID NO: 15)B-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27 29SLREL-LS*N-LGDAGV-LLCEGLL-P-C where * is N or S (SEQ ID NO: 16)

Finally, for a threshold of 25% or more identical amino acids at a givenposition the RI repeat motif pair was almost completely defined by onlyone amino acid per position.

A-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27+LEKLWLEDCGLTAAGCKDLCSVLRANP where + is R or K (SEQ ID NO: 17)B-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27 29SLRELDLS*NELGDAGVRLLCEGLL#PGC where * is N or S (SEQ ID NO: 18) and #is D or Q

This is to illustrate how a sequence motif can be derived only fromsequence information and alignment. However, preferably structuralinformation should be taken into account

2) Defining Framework and Target Interaction Residue Positions

The analysis of both A- and B-type LRR units revealed that the sidechains of the amino acids at positions 2, 5, 7, 10, 12, 17, 20, and 24are always oriented towards the hydrophobic core (Kobe and Deisenhofer,1994 and FIGS. 4b and 4d ) and these amino acids constitute a subset ofthe framework residues. Other framework residues are the glycine atposition 16 and the prolines at position 28 in the A-type LRR unit(abbreviated A28) and position 27 in the 8-type LRR unit (abbreviatedB27), since they initiate and terminate the α-helix of each LRR unit.Furthermore, positions A1, A3, A13, A18, A19, A22, A25, A27 and B1, B3,B11, B14, B18, B22, and B26 most often harbour hydrophilic amino acidresidues oriented towards the surrounding solvent and were treated asframework positions. Similarly, positions A14, A15, A21, A23, A26, andB15, B19, B21, B25, and B29 are usually occupied by hydrophobic aminoacid residues stabilising the interface of the repeat modules and arethus also treated as framework positions. Further, positions A11, B13,B23 and B28 feature glycine with allow more flexibility than other aminoacids and are therefore also important for the framework. In contrast,the positions 4, 6, 8, and 9 were defined to be the target interactionpositions in the RI repeat motif pair.

3) Replacing Unfavorable Amino Acids

The RI consensus is also characterised by extremely well conservedcysteines at positions A10 and A17 and positions B21 and B29. However,as free cysteines may be oxidised and cause complications, it isdesirable to design cysteine free modules. Therefore, appropriatereplacements were sought. Inspection of the three-dimensional structure(MTS#1) revealed that the cysteine at position A10 made a H-bond.Further, alignments to more distant LRR molecules revealed the presenceof either asparagine, serine, or threonine in most cases. Thus, theposition A10 in the LRR module was designed to be occupied by thesethree amino acids. Similarly, position A17 was found to be part of thehydrophobic core, which is why in the LRR module methionine, leucine, orvaline were used. At the same time, these two positions A10 and A17constitute cases where framework positions are randomised. At positionB21, the cysteine of the first and last repeats in all analysed RIsequences was constantly occupied by leucine (with one exception ofvaline) and thus defined to be leucine in the final LRR module. In caseof position B29, the choice was accordingly between serine andthreonine, where the threonine was chosen to allow an assembly with therestriction endonuclease sites of BssHII and MluI (for a detaileddescription see FIG. 6).

The last remaining cysteine, which occurred in 36% of the analysedposition A21 (FIG. 5c ), was set to be alanine because this was thesecond most frequent amino acid at the given position and also seemed tomatch the hydrophobic environment. The decision was facilitated since itwas noted that in most cases where leucine was found at position B21,position A21 was occupied by alanine. In other words, the leucine atposition B21 seems to prefer alanine at position A21. Thus, stacking wasbelieved to be supported best with this choice in the LRR module.

Another decision was required for position A1. From the two possiblepositively charge amino acids lysine and arginine, the latter was chosento match the above mentioned restriction endonuclease sites.

The refined repeat sequence motif can thus be described by the followingsequence

A-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27RLEKLWLED2GLTAAG4KDLASVLRANP where 2 is N or S or T (SEQ ID NO: 19)and 4 is L or M or V B-type LRR consensus1  3  5  7  9  11 13 15 17 19 21 23 25 27 29SLRELDLS*NELGDAGVRLLLEGLL#PGT where * is N or S (SEQ ID NO: 20) and #is D or Q4) Defining the Target Interaction Residues

For the definition of the target interaction positions, both the humanRI-angiogenin (Papageorgiou of al., 1997) and the pig RI-RNase A (Kobeand Deisenhofer, 1995) complexes were analysed. Apart from extensiveinteractions at both the N- and the C-terminal capping units, theinteractions of repeat units involved most frequently positions 6, 8,and 9 of the A-type LRR unit, whereas in the B-type LRR unit, positions4, 6 and 9 were used most often. All these positions are characterisedby side chains emanating from the β-strand of the LRR unit (FIG. 4e )and are therefore suited for target interactions. As however, theglutamate at position 4 of the B-type LRR unit was present withoutexception and an additional structural importance could not bedismissed, we refrained from randomising this position. Thus, thisposition constitutes a case where a target interaction position is notrandomised. In contrast, the position A4 was also defined to berandomised since it showed less than 30% conservation. Thereforepositions A4, A6, A8, and A9 and positions B6 and B9 were randomised inthe LRR module. The chosen subset of the amino acids at the randomisedpositions largely reflected the physicochemical properties of naturallyoccurring ones and therefore all charged and some H-bond forming andaromatic amino acids known to support binding in many instances werechosen. At the same time, the decision was taken to allow larger aminoacids only in the A-type LRR unit positions and only smaller ones at theB-type LRR unit positions minimising steric hindrance in an alternatingcontext. Thus, the obtained repeat sequence motif at this stage can bedescribed as follows

A-type LRR consensus 1  3  5  7  9  11 13 15 17 15 21 23 25 27RLE1L1L112GLTAAG4KDLASVLRANP where 1 is D, E, N, Q, S, R, K, W, Y(SEQ ID NO: 21) and 2 is N or S or T and 4 is L or M or VB-type LRR consensus 1..3  5  7  9  11 13 13 17 19 21 23 25 27 29SLREL3LS3NELGDAGVRLLLEGLL#PGT where 3 is G, S, D, N, H or T(SEQ ID NO: 22) and # is D or Q

Thus, randomisation at eight positions resulted in 2.8×10⁵ independentRI repeat module pairs. In other words, the synthesis of moleculessatisfying the above described repeat sequence motif will create about300000 independent but highly homologous members.

Another position analysed in detail is in the loop region on top of bothLRR repeat units, namely position 11. Since the consensus in both A-typeand B-type LRR unit was 36% and 25% respectively, the occurrence ofpairs of amino acids was checked. In the B-type LRR unit, charged aminoacids were slightly preferred at position 11 and a lysine often occurredwith an aspartate in the A-type LRR unit. This putative salt bridge wasbelieved to increase stability and solubility of the designed LRR moduleand was therefore chosen. Another possibility (glycine A11 and glutamateat B11) was dismissed for fear of too high flexibility.

The choice at position A14 was between alanine and glutamate, where thelatter was again chosen to enhance the solubility and the correctorientation the hydrophilic outer shell. Similarly, the position B22suggested either glutamate or glutamine, where the latter Was chosensince it seemed to better match the serine at A22 defined previously.

Finally, position 26 was subject to scrutiny, where the choice wasbetween alanine at A26 together with glutamine at B26 on the one hand,and serine at A26 together with aspartate at B26. The latter variant wasadopted to again enhance the solubility of the LRR module.

Thus, the RI repeat motif pair looks as follows (alterations are printedbold)

A-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27RLE1L1L112DLTEAG4KDLASVLRSNP where 1 is D, E, N, Q, S, R, K, W, Y(SEQ ID NO: 23) and 2 is N or S or T and 4 is L or M or VB-type LRR consensus 1  3  5  7  9  11 13 15 17 19 21 23 25 27 29SLREL3LS3NKLGDAGVRLLLQGLLDPGT where 3 is G, S, D, N, H or T(SEQ ID NO: 24)5) Designing a Repeat Domain Derived from the LRR of Mammalian RI

Assembling multiple repeat modules into a domain is straightforward.Here, we undertook an approach involving two different restrictionenzymes creating compatible overhangs (cf. FIG. 6). Thus, the directionof the ligation can simply be controlled by redigesting the ligationproducts, where only correctly ligated molecules are not cut.

Additionally, we chose to complement the assembled LRR modules by N- andC-terminal capping modules designed to shield the putative jointhydrophobic core of the repeat domain from the surrounding solvent. Theanalysis of the mammalian RI proteins revealed that the first and thelast LRR units differed significantly from the consensus described above(FIGS. 5c and 5d ). For simplicity, the corresponding capping units ofthe human RI were cloned with slight modifications and are henceforthreferred to as capping modules. Thus, amino acids 1 to 28 for theN-terminal capping module and amino acids 427 to 460 of human RI for theC-terminal module were used, and a short linker encoding the amino acidresidues PYAR (SEQ ID NO: 75) was introduced between the N-terminalcapping module and the RI repeat module pairs to match the lengthrequirements.

When the devised amino acid consensus was reverse translated into a DNAsequence the following parameters were taken into account: No undesiredrestriction enzyme recognition sequences were allowed within the repeatmodule pair and the codon usage was optimised for expression in E. coli.

6) Preparation of the Expression Plasmids

To obtain the N-terminal module flanked by appropriate restrictiondigestion sites, the DNA of pTRP-PRI (Lee and Vallee, 1989) wasamplified with oligonucleotides MTS2 and MTS4 (Table 1) givingPCR-fragment N. Thus, at the 5′-end, an NcoI and a BamHI wereintroduced, whereas the 3′-end featured a BssHII and a HindIII site. Theresulting DNA fragment is shown with translated amino acids in thecorrect frame (above the boxed part in FIG. 8).

The PCR-fragment N was ligated into the NcoI and HindIII sites of pTFT74(Ge et al., 1995) yielding plasmid pTFT_N (FIG. 9a ). At the same time,an N-terminal Flag-tag and a 6×His-tag (SEQ ID NO: 76) were introduced.Several vectors were derived from pTFT_N for the insertion of the abovedescribed repeat modules. The NcoI-HindIII insert of pTFT_N was clonedinto pQE60 (QIAgen, Hilden, Germany) prepared with the same restrictiondigestion enzymes (giving plasmid pQE_N). The BamHI-HindIII insert ofpTFT_N (that is without N-terminal Flag-tag and the 6×His-tag) (SEQ IDNO: 76) was cloned into pQE60 derivative downstream of the lambda phageprotein D gene insert in frame to yield a C-terminally fused repeatdomain (giving plasmid pQE-pD_N).

The pTFT derivatives feature a T7 polymerase promoter under a lacoperator, whereas the pQE derivatives offer a T5 polymerase promotorunder the same control system. Lambda phage protein D as N-terminalfusion partner was chosen to increase the solubility and expression(Forrer and Jaussi, 1998).

7) Synthesis of the Repeat Module Libraries

Oligonucleotides MTS7 and MTS9 were partly assembled from trinucleotides(Virnekäs et al., 1994) all other oligonucleotides were synthesised withstandard techniques.

The strategy presented below describes a way to obtain polymers of DNAfragments in a defined direction using palindromic restriction enzymesand ligation. One such possibility is to use the restriction enzymesBssHII and MluI (FIG. 6) which create compatible overhangs. If DNAfragments with the same overhang but different original recognitionsites are religated a new combined site (named * in FIG. 6 and FIGS. 7ato 7c ) will be formed which cannot be digested by either of theoriginal enzymes (FIG. 6). However, the ligation of identical ends leadsto the original recognition site and these molecules can therefore bedistinguished by restriction digestion. Other pairs of restrictionenzymes with compatible overhangs are well known to those skilled in theart.

The following step numbering refers to the one used in FIG. 7a to c.

(Step I) To obtain the first library of repeat modules the partlyrandomised oligonucleotides MTS7, MTS8, MTS9, and MTS10 were assembledby PCR and were amplified with a 10-fold molar excess of MTS11 b andMTS14b in one step (95 degrees for 2 min; then 20 cycles of 95 degreesfor 15 sec 55 degrees for 15 sec, and 72 degrees for 20 sec followed by72 degrees, 1 min). In case of the LRR library described here, theinitial PCR assembles the above described A/B pair into one module. Theresulting DNA fragment is shown with translated amino acids in thecorrect frame (boxed part in FIG. 8, the oligonucleotides are shown asarrows).

(Step II) Separate extensive restriction digestion with either BamHI andMluI or BssHII was followed by ligation with T4 ligase (1 hour at roomtemperature and neat inactivation of the enzyme). The resulting ligationproduct was purified by low melting point agarose gel electrophoresis.The band corresponding to the dimer repeat module was isolated and theDNA was recovered after β-agarase digestion by ethanol precipitation.

(Step III) To amplify the dimer of repeat modules a second PCR reactionwith primers T7pro and srpTFT1 (95 degrees for 2 min; then 15 cycles of95 degrees for 15 sec; 50 degrees for 15 sec, 72 degrees for 40 secfollowed by 72 degrees for 1 min) was performed. In case of the LRRlibrary this step yielded two NB pairs, that is four leucine-richrepeats. As 1 microgram template corresponding to about 10¹² moleculeswas used for the LRR library the total theoretical diversity was stillcovered at this stage.

(Step IV) For the tetramer, the obtained DNA was again digested witheither BamHI and MluI or BssHII. For longer polymers mixtures of singleand doubly digested DNA fragments were prepared.

(Step V) The ligation, restriction digestion, and purification can berepeated until the desired number of repeat modules is obtained.

(Step VI) To obtain a DNA fragment with two different non-compatiblerestriction digestion sites at both ends for the directed and efficientcloning into a plasmid, the following “capping” strategy was devised.The C-terminal repeat unit of human RI was also amplified from plasmidpTRP-PRI by PCR thereby introducing a BssHII restriction site on the5′-end and a HindIII restriction site at the 3′-end. The resulting DNAfragment is shown with translated amino acids in the correct frame(below the boxed part in FIG. 8).

The primers MTS5a and MTS3 were used in this PCR reaction (95 degreesfor 2 min, then 20 cycles of 95 degrees for 15 sec, 45 degrees for 15sec, and 72 degrees for 10 sec followed by 72 degrees for 1 min) and theproduct was QIAquick purified and restriction digested with BssHII.

(Step VII) The BssHII digested C-terminal repeat module was ligated to aMluI digested polymers by T4 ligase (1 hour at room temperature and heatinactivation of the enzyme). The subsequent extensive restrictiondigestion with BssHII. MluI, and HindIII ascertained the correctorientation of the modules. The mixture was separated by low meltingpoint agarose gel electrophoresis and the desired bands were recoveredas above. Finally, the recovered fragments were ligated into any of theBssHII-HindIII digested plasmids pTFT_N, pTFT-pD_N, pQE_N or pQE-pD_N.The resulting ligation mix was QIAquick purified and used forelectroporation of XL10Gold cells prepared according to Sidhu et al.(2000).

The above described protocol results in different libraries of plasmidsand two representative diagrams of such plasmids are shown (FIGS. 9b and9c ),

8) Characterization of the Repeat Module Protein Libraries

Standard DNA sequencing techniques were used to determine the DNAsequence of the expression plasmids. As an example the DNA sequence ofclone D17 (compare expression in FIGS. 11c, 11d, and 12d ) is given(FIGS. 10a and 10b ). The N-terminal module and the four repeat modulesas well as the C-terminal module are indicated.

Expression was essentially performed as described (QIAgen“QIAexpressionist”) and the soluble and insoluble proteins of singleclones and/or pools of clones after sonification were separated bySOS-PAGE analysis and Coomassie stained (FIG. 11a-d ). Western blotanalysis was performed according to the protocol supplied by themanufacturer. Antibody anti-Flag M2 (Sigma) was used for the constructswithout N-terminal protein D, whereas anti-RGS-His (Qiagen) was used forconstructs with N-terminal protein (FIG. 12a-d ).

Purifications (FIGS. 13 and 14), CD spectrometry (FIG. 15) and sizeexclusion chromatography (FIG. 16) were carried out as described inexample 2.

9) Selection of (Poly)Peptide/Proteins which Inhibit Bacterial Toxins

Various bacterial toxins are known to occur together with thecorresponding antitoxin because even a moderate level of toxin alonecannot be tolerated in bacteria. Therefore, the gene of CcdB (Jensen etal., 1995) was cloned into a low copy plasmid of the pZ series with atightly repressed tetracyclin promotor in a tetracyclin repressor strainlike DH5□Z1 (Lutz and Bujard, 1997), XL10Gold or XL1Blue. In parallel,wild-type barnase (Hartley, 1988) and the barnaseH102K mutant with 0.1%activity (Jucovic and Hartley, 1996) were cloned. Chemically competentcells with one of these toxin plasmids were prepared as described (Inoueet al., 1990) and electroporation competent cells harbouring one ofthese toxin plasmids were prepared as described (Sidhu et al., 2000).For the selection of plasmids encoding a toxin inhibitor cells weretransformed with the LRR-based library, plated on selective plates (LBmedium supplied with 50 mg/L ampicilin, 20 mg/L kanamycin, 40 micromolarIPTG, and 30 microgram/L anhydrotetracyclin), and grown at either 25 or37° C. To confirm that inhibitory properties are plasmid-linked, the pQEderivatives were reisolated and retransformed.

Screening for Efficiently Folding Constructs

GFP has been successfully used as a folding reporter when fused to theC-terminus of the target protein (Waldo et al., 1999). Rapidlyaggregating targets do not allow folding of C-terminally fused GFP andcolonies can be screened in UV light. The fluorescence of GFP correlatedwith the amount of correctly folded protein. In our strategy. GFP wascloned into the NheI and EcoRI sites designed at the C-terminus obtainedby PCR amplification using MTS5a and MTS6 and again pTRP-PRI astemplate. Hereby, a 12 amino acid linker GSAGSAAGSGEF (SEQ ID NO: 25)was introduced. The resulting DNA fragment is shown with translatedamino acids in the correct frame (at the bottom in FIG. 8).

Selection for Constructs without Stop-Codons

To reduce the number of frameshifts and stop-codons after theconstruction of the library, the constructs were cloned upstream of alinker connecting to the chloramphenicol resistance gene and viableclones were selected on plates.

Selection for Binding Targets Using Display Techniques

To identify binding partners in vitro, both ribosome display (Hanes etal., 1998) and phage display (Dunn, 1996) was used. Binding partnerswere RNase A and Onconase (Wu et al., 1993b) from the RNase superfamilyand protein D (Forrer and Jaussi, 1998), an unrelated small polypeptide.

Selection for Binding Targets Using the Protein Complementation Assay

To identify binding partners, an E. coli genomic library was fused tothe DHFR1 fragment (Pelletier et al., 1998), whereas the LRR-basedlibrary was fused next to DHFR2. Selection on M9 plates containingtrimethoprim lead to interacting molecules.

DNA Module Shuffling for the Improvement of the Obtained Constructs

For further evolutionary improvements, the obtained constructs weresubjected to DNA shuffling (Stemmer, 1994) and back-crossing. Thus,improvements could be enriched and mutations without effect were lost.

Example 2

Collection of (Poly)Peptide/Proteins Comprising Repeat Modules Derivedfrom Ankyrin Repeat Units

A method for the generation of designed ankyrin repeat proteinsaccording to the present invention is described. The method allows theconstruction of ankyrin repeat proteins of various length by using anN-terminal ankyrin capping module, two or several ankyrin repeat modulesand a C-terminal ankyrin capping module.

The definition of the ankyrin repeat motif which was the basis for thegeneration of a collection of ankyrin repeat modules in EXAMPLE 1 isdescribed below. The analysis leading to the ankyrin repeat motifincluded search of public databases for naturally existing ankyrinrepeat proteins as well as structural analysis of ankyrin repeatproteins with known three-dimensional structure. By way of thisanalysis, a sequence motif for the ankyrin repeat modules was derivedand ankyrin capping modules were derived. Furthermore, the positions offramework and target interaction residues were determined for theankyrin repeat motif. To generate a library of ankyrin repeat modules,17 out of 20 natural amino acids were allowed at the positions of targetinteraction residues in the ankyrin repeat motif. The positions of theframework residues were specified to certain amino acids each. Theresulting peptide sequences were reverse translated such that the codonusage was optimal in Escherichia coli but did not create unwantedrestriction sites. Oligonucleotides were designed to allow assembly PCRof the ankyrin repeat modules. Trinucleotide oligonucleotides (Virnekäset al., 1994) as well as conventional oligonucleotides were used (Tables2 and 3). Similarly, the N- and C-terminal ankyrin capping modules weregenerated by assembly PCR using conventional oligonucleotides (Table 2).The resulting PCR products all contained type IIs restriction enzymerecognition sites (FIG. 17) at those ends that subsequently would beconnected to the DNA of the next/previous repeat- (or capping) module(FIG. 18). When cut by the respective restriction enzymes, the generatedcompatible ends of the modules could be ligated in frame in aunidirectional way. Hence, the N-terminal ankyrin capping module couldbe ligated to one or several ankyrin repeat modules and the ligationproducts could be Heated to the C-terminal ankyrin capping module. Asthe DNA differed in defined positions, the method allowed thesimultaneous assembly of a diverse set of DNA molecules encoding acollection of ankyrin repeat proteins. Members of the resultingcollections of ankyrin repeat proteins were characterised by expression,purification, circular dichroism spectroscopy, denaturation experiments,size exclusion chromatography as well as crystallisation. Theexperiments demonstrated that unselected members of this ankyrin repeatprotein library can be expressed in the reductive environment of thecytoplasm at high levels in a soluble and folded conformation.

Definition of the Ankyrin Repeat Motif Sequence

PROCEDURE and RESULT: The ankyrin repeat motif used as an example forthe present invention was derived from ankyrin repeat protein sequenceanalysis as well as from structural analysis of ankyrin repeat proteinswith known three-dimensional structure (date: August 2000).

The SMART database (Schultz et al., 2000) was first searched for aminoacid sequences of ankyrin repeat units. A Clustal-W (Thompson et al.,1994) alignment of 229 ankyrin repeat units served as template for thedetermination of an ankyrin repeat unit consensus “A” (FIG. 19).Consensus “A” was determined by calculation of the residue-frequencyoccurrence for each position of the alignment of ankyrin repeat units.The 229 ankyrin repeat units considered did not contain inserts ordeletions compared to a previously stated general ankyrin repeat unitconsensus sequence (Sedgwick and Smerdon, 1999). Consensus “A”, however,included only residues 3 to 32 (FIG. 19) of the 33 amino acids longconsensus sequence of Sedgwick and Smerdon (1999). To further refine theconsensus and define the lacking positions, a BLAST (Altschul et al.,1990) search against GenBank (Benson et al., 2000) was performed usingdefault parameters. For this search, consensus “A” was submitted in acircularly permutated form with position 20 as first amino acid (FIG.19). The missing or ambiguous positions were filled with residues thathad highest frequency in a consensus of ankyrin repeat units of ankyrinrepeat proteins with known three-dimensional structure (manuallyaligned, statistics as described above). The first 200 of the resultingBLAST hits were manually aligned and the ankyrin repeat unit consensus“A” was refined by residue frequency analysis as stated above yieldingconsensus “B” (FIG. 19). Consensus “B” was confirmed by an identicalanalysis of the pfam database (Bateman et al., 1999; data not shown).

The final ankyrin repeat unit consensus “C” (FIG. 19) was obtained byintegration of the methods mentioned in this paragraph. Publishedthree-dimensional structures of ankyrin repeat proteins were visuallyinspected to further decide which amino acids were optimal at a certainposition. The three-dimensional structure showing highest homology toankyrin repeat unit consensus “B”, the mouse GA-binding protein beta 1subunit (AC: 2981726, pdb; 1AWC; Batchelor et al., 1998), was theguideline in most instances, but other structures such as human p18 (AC:4139830, pdb: 1IHB; Venkatamarani et al., 1998) were also considered.The mutual dependence of pairs, triplets and quatruplets of amino acidsin naturally occurring ankyrin repeat unit sequences was also used tofurther develop or assure consensus “B”. Furthermore, modelingapproaches (insightII package; Informax Inc., USA) including homologymodeling and energy minimisations have been performed and the consensussequence was developed towards optimal cavity avoidance and packingoptimisation. It was further ensured that the secondary structurepropensity (O'Neil and DeGrado, 1990; Chou and Fasman, 1978) of eachresidue of the consensus matched the secondary structure at thecorresponding position in natural ankyrin repeat units. In addition, thesecondary structure was analysed and verified using PhD-prediction(Rost, B., 1996). Protein stability and protease resistance of theconsensus was then analysed using PEST (Rogers et al., 1986; SwissInstitute of Bioinformatics. Switzerland) and peptidesort of GCG(Accelrys, USA; Womble, D. D., 2000) and the consensus was predicted tobe sufficiently stable.

Critical residues during the definition of ankyrin repeat unit consensus“B” (FIG. 19) to consensus “C” (FIG. 19) were positions 16, 17, 18, 19,21, 22, 25 and 26. Position 16 was finally determined to be a histidine,since it makes buried H-bonds from its position to the previous repeat.The leucine at position 17 was finally preferred to other amino acidssince it stabilises the interface of two repeat modules. The glutamateof position 18 was chosen as repeated glutamates and aspartates occur inhuman p18 at this position. Similarly, the glutamate at position 21occurs in multiple successive copies in mouse GA-binding protein. Lysine25 was preferred to other amino acids as the basic residues arginine andlysine occur repeatedly in mouse GA-binding protein as well. Forposition 26, the compromise of taking any of the three amino acidshistidine, tyrosine or asparagine was chosen, as these amino acids allfulfil the requirements for this position. Accordingly, the positions 19and 22 were occupied by isoleucine or valine and valine or leucine,respectively, as these residues fitted equally well.

The finally determined ankyrin repeat unit consensus “C” (FIG. 19)served as basis for the ankyrin repeat modules. The sequence of theankyrin repeat motif is shown in FIG. 20. For cloning reasons the motifis based on a circularly permutated consensus “C”. In order to match theconsensus numbering scheme used in FIG. 19 and used by Sedgwick andSmerdon (1999), the numbers of the positions in the ankyrin repeat motifwere circularly permutated in parallel to the amino acid sequence. Theankyrin repeat motif has a length of 33 amino acids, whereof 27positions were defined to be framework residues and 6 positions weredefined as target interaction residues. The positions of frameworkresidues were defined using ankyrin repeat unit consensus “C”. Analysesof three-dimensional structures showed that positions 2, 3, 5, 13, 14and 33 of the ankyrin repeat units are often involved in protein-proteininteractions and hence constitute the target interaction residues. Thiswas also suggested by the high variability these positions showed duringankyrin repeat unit consensus definition. For the ankyrin repeatmodules, these residues were defined to be any of the 17 amino acids A,D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

Thus, the number of independent members of the collection of ankyrinrepeat modules can be calculated to be 3·17⁶=72′412′707.

Definition of the Ankyrin Capping Modules

PROCEDURE and RESULT. As the derived ankyrin repeat motif showed highhomology to the beta 1 subunit of the mouse GA-binding protein (GABPbeta 1; AC: 2981726; Batchelor et al., 1998), the N- and C-terminalankyrin repeat capping units (repeats 1 and 5 according to Batchelor etal., 1998) of the latter protein were chosen as a basis for the N- andC-terminal capping modules. Both the N- and C-terminal ankyrin cappingmodule had to be changed compared to the mouse GA-binding protein beta 1capping units. The N-terminal GA-binding protein beta 1 capping unit wasmodified in its loop to sterically fit the design of the ankyrin repeatmotif. The C-terminal GA-binding protein beta 1 capping unit wasmodified at several positions. Parts of the loop of repeat 4 and thebeta hairpin connecting repeat 4 and 5 of GA-binding protein beta 1(Batchelor et al., 1998) had to be included into the C-terminal cappingmodule for cloning reasons. Thereby, the loop and the beta hairpin weremodified to sterically fit the design of the ankyrin repeat motif. Themodifications can be seen in FIG. 21, where GABP beta 1 is aligned toE3-5, a member of a protein library according to the present invention(see below).

Experimental Procedures

For all following sections of EXAMPLE A, techniques were performedaccording to protocols as described in Sambrook, J., Fritsch, E. F. andManiatis, T. (1989; Molecular cloning: a laboratory manual. Cold springlaboratory press, New York) or in volumes 1 to 4 of Ausubel, F. M.,Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.and Struhl, K. (1994; Current protocols in molecular biology. John Wileyand Sons, Inc., New York) or in volumes 1 and 2 of Coligan, J. E., Dunn,B. M., Ploegh, H. L. Speicher, D. W. and Wingfield, P. T. (1995; Currentprotocols in protein science. John Wiley and Sons, Inc., New York).

Synthesis of DNA Encoding Ankyrin Repeat Proteins According to thePresent Invention

PROCEDURE and RESULT: Oligonucleotides INT1 and INT2 were partlyassembled from trinucleotides (Virnekäs et al., 1994) and were obtainedfrom MorphoSys (Germany). All other oligonucleotides were synthesisedwith standard techniques and were from Microsynth (Switzerland, cf.Tables 2 and 3). Oligonucleotides for amplification of DNA were used at100 μM stock concentration, while the ones used as templates were usedas 10 μM stock. Enzymes and buffers were from New England Biolabs (USA)or Fermentas (Lithuania). Cloning strain was E. coli XL1-Blue(Stratagene).

The ankyrin repeat modules were generated by assembly PCR usingoligonucleotides (1 μl each) INT1, INT2, INT3, INT4, INT6 and INT6a [5min 95° C., 20·(30 sec 95° C., 1 min 50° C., 30 sec 72° C.), 5 min 72°C.] and Vent DNA polymerase in its standard buffer supplemented withadditional 3.5 mM MgSO₄ in a final volume of 50 μl.

The N-terminal ankyrin capping module was prepared by assembly PCR usingoligonucleotides (1 μl each) EWT1, EWT2, TEN3 and INT6 [5 min 95° C.,30·(30 sec 95° C., 1 min 40° C., 30 sec 72° C.), 5 min 72° C.] and VentDNA polymerase in its standard buffer in 50 μl reaction volume. Theresulting DNA was cloned via BamHI/HindIII into pQE30 (QIAgen, Germany).The DNA sequence was verified using standard techniques. The C-terminalankyrin capping module was prepared accordingly, but by usingoligonucleotides WTC1, WTC2, WTC3 and INT5.

The ligation of the DNA encoding an ankyrin repeat protein from singleankyrin repeat modules and ankyrin repeat capping modules is representedschematically in FIG. 18. To assemble ankyrin repeat proteins, thecloned N-terminal ankyrin capping module was FOR-amplified usingoligonucleotides TEN3 and INT6a (conditions as above for the N-terminalankyrin capping module). The DNA was purified using the QIAquick DNApurification kit (QIAgen, Germany), cut with BsaI and repurified usingthe same kit. The N-terminal ankyrin capping module was then ligatedonto BplI cut and purified ankyrin repeat module. This directionalcloning was possible since the cutting sequences BplI and BsaI, two typeIIs restriction enzymes which recognise a DNA sequence different fromthe cutting sequence (FIG. 17), was chosen to be asymmetric butcompatible with each other. The ligation product, termed N1, wasgel-purified (LMP-agarose, β-agarase, sodiumacetate/ethanolprecipitation) and PCR-amplified using oligonucleotides (1 μl each) EWT3and INT6b [5 min 95° C., 20·(30 sec 95° C., 30 sec 50° C., 30 sec 72°C.), 5 min 72° C.] and Vent DNA polymerase in its standard buffer in 50μl reaction volume. The amplified product was purified using QIAquick,cleaved with BsaI and purified again. The subsequent ligation to BplIcut ankyrin repeat modules started a new cycle of elongation which wasrepeated until the desired number of ankyrin repeat modules was added tothe N-terminal ankyrin capping module (termed N2, N3, N4 etc.). DNApieces corresponding to FOR-amplified N2, N3 and N4 were then cut withBsaI and ligated to a previously BplI-cut FOR product of the clonedC-terminal ankyrin capping module. This yielded DNA molecules encodingN2C, N3C and N4C ankyrin repeat protein libraries. The final productswere FOR amplified using 1 μl of each EWT3 and WTC3 each [5 min 95° C.,25·(30 sec 95° C., 30 sec 50° C., 1 min 72° C.), 5 min 72° C.] andcloned via BamHI/HindIII into pQE30 (QIAgen).

Protein Expression and Purification

PROCEDURE: E. coli XL1-Blue (Stratagene) was used as strain for theexpression of ankyrin repeat proteins of different lengths. Two clonescorresponding to N2C (named E2-5 and E2-17), two clones corresponding toN3C (E3-5 and E3-19) and two clones corresponding to N4C (E4-2 and E4-8)were randomly chosen and analysed further. 25 ml of stationary overnightcultures (LB, 1% glucose, 100 mg/l ampicillin; 37° C.) of these cloneswere used to inoculate 1 l cultures (same media as preculture). AtOD₆₀₀=0.7, the cultures were induced with 300 μM IPTG and incubated forfour hours. Samples were taken at various timepoints and analysed viaSDS-PAGE (see FIG. 22). The cultures were centrifuged and the resultingpellets were taken up in 40 ml TBS₅₀₀ (50 mM TrisHCl, pH 8.0, 500 mMNaCl) and sonified. Then the lysates were supplemented with 10%glycerole and 20 mM imidazole and recentrifuged. The resultingsupernatant was used for purification over a His-tag column (2.5 clcolumn volume) according to the manufacturer (QIAgen, Germany).

RESULTS: Cell fractionation experiments showed that all ankyrin repeatproteins were soluble expressed with yields of 200 mg/l culture (FIG.22), His-tag purification led to pure protein in a single purificationstep (FIG. 23). The proteins integrity was further confirmed by massspectroscopy (not shown). The soluble expression indicates properfolding of the designed repeat proteins.

Size Exclusion Chromatography

PROCEDURE: The six purified samples described above were analysed on aSuperdex 75 column (Amersham Pharmacia Biotech, USA) using a PharmaciaSMART system at a flow rate of 60 μl/min and TBS 150 (50 mM TrisHCl, pH7.5; 150 mM NaCl) as running buffer. Standards were □-amylase (Sigma)and the phage proteins pD and SHP (Yang et al., 2000). As an example theelution profile of a N3C-library member, E3-5, is shown in FIG. 24.

RESULTS: The elution profile showed that the proteins investigated werein most cases exclusively monomeric, while a minor number of proteinsamples (E2-17 and E4-8) showed multimerised, but soluble species inaddition to the monomers. The retention measured by gel filtrationindicated that the investigated proteins are folded and not randomcoils.

CD Spectroscopy

PROCEDURE: The circular dichroism spectra of a randomly chosen ankyrinrepeat protein generated according to the present invention (E3-5, a N3Cmolecule) were recorded either in 10 mM sodium phosphate buffer pH 6.5(native) or 20 mM sodium phosphate buffer pH 6.5 and 6 M Guanidiniumhydrochloride (denatured) using a Jasco J-715 instrument [Jasco, Japan;10 nm/s, 8 sec response, 0.2 nm data pitch, 2 nm band width, 195-250 nm(native) or 212-250 nm (denatured), three accumulations, measurements intriplicates, 1 mm cuvette]. The CD signal was converted to mean residueelipticity using the concentration of the sample determinedspectrophotometrically at 280 nm under denaturing conditions.

RESULTS: E3-5 shows an alpha-helical spectrum under native conditionswith minima at 208 nm and 222 nm. The secondary structure is lost in 6 MGuanidinium hydrochloride (FIG. 25). This indicates the proper formationof secondary structure elements in E3-5.

Denaturation Behaviour

PROCEDURE: The denaturation behaviour of randomly chosen ankyrin repeatproteins generated according to the present invention (E2-6. E3-5 andE4-8. FIG. 22) was measured via circular dichroism spectroscopybasically as indicated in FIG. 25 but using different buffers.Guanidiniumhydrochloride denaturation curves were measured by CDspectroscopy at 220 nm using the different proteins incubated indifferent concentrations of guanidinium hydrochloride in 20 mM NaPO₄p116.5, 100 mM NaCl, overnight at room temperature. The circulardichroism signal at 220 nm was measured for each sample in triplicates.

RESULTS: The denaturation curves of E2-5. E3-5 and E4-8 againstdifferent concentrations of guanidinium hydrochloride are shown in FIG.26. The midpoint of denaturation is in a range of 2.5 to 3.8 Mguanidiniumhydrochloride. Hence, the secondary structure is lost only athigh concentrations of denaturing agent indicating a relatively highstability of the investigated molecules.

Crystallisation

PROCEDURE and RESULT: The ankyrin repeat protein E3-5, a N3C librarymember according to the present invention, was crystallised in 20% PEG6000, 100 mM MES/NaOH pH 6.0 in five days at 20° C., hanging droplet (2μl protein and 2 μl buffer mixed; 500 μl buffer reservoir) from asolution of 9 mg Protein per ml in TBS 50 (50 mM TrisHCl, pH 8.0, 50 mMNaCl: cf. FIG. 27). The crystal refracted to 3 Å in preliminary X-rayexperiments (not shown).

Tables

Table 1: Oligonucleotides used for the cloning of the library derivedfrom human RI (SEQ ID NOS 26-37, respectively in order of appearance,peptide (SEQ ID NO: 77 and 6×-His tag (SEQ ID NO: 76);

Table 2: Oligonucleotides used for the generation of ankyrin repeatmodules according to example 2 (SEQ ID NOS 38-43, respectively in orderof appearance);

Table 3: Oligonucleotides used for the generation of the N- andC-terminal ankyrin capping modules as well as for the cloning of ankyrinrepeat proteins containing more than one ankyrin repeat module (SEQ IDNOS 44-52, respectively in order of appearance).

TABLE 1 Oligonucleotides used for the cloning of the libraryderived from human RI (SEQ ID NOS 26-37, respectively in order ofappearance, peptide (SEQ ID NO: 77 and 6X-His tag (SEQ ID NO: 76)Sequence in 5′-3′ direction Name (restriction sites)¹ Description MTS2CATGCCATGGACTACAAGGATCATCACCATCACCATCACG fwd2 PCR primer to obtainGATCCctggacatccag (NcoI, BamHI) human RI with initial Flag-tag MDYKD and 6xHis-tag MTS4 GCATAAGCTTATCACTCGAGGCGCGCGTAGGGctgctggarev² PCR primer to obtain gcagagg (HindIII, XhoI, BssHII)N-term. RI unit MTS3 GCATAAGCTTATCAggagatgaccc (HindIII)rev² PCR primer to obtain human RI MTS5aCATGCCATGGGcgcgCctcgagcagctggtcc (NcoI, fwd² PCR primer for newBssHII, XhoI) C-term. unit MTS7 TTGGCGCGCCTGGAGNNNCTGNNNCTGNNNNNNNNNgaccfwd² assembly left, 4 tcaccgaggccggc (BssHII)³ library elements, 1 codonfor S, N, T MTS8 ccgcaggctcgggttggaGCGGAGCACGCTGGCCAGGTCCrev² assembly left, 1 TTCANgccggcctcggtgaggtc codon for L, M, V MTS9tccaacccgagcctgcggGAGCTGNNNCTGAGCNNNaaca fwd² assembly right, 2agctcggcgatgca library elements MTS10CCGCTCGAGACGCGTGCCGGGGTCCAGCAGCCCCTGCAAG rev² assembly rightAGCAGCCGCACGCCtgcatcgccgagcttgtt (XhoI) MTS11bTAATACGACTCACTATAGGGttggcgcgcctggag fwd² PCR primer to amplify (BssHII)the assembly MTS14b GGCTTTGTTAGCAGCCGGATCctcgagacgcgtgccggggtrev² PCR primer to amplify c (BamHI, XhoI, MiuI) the assembly T7proAAATtaatacgactcactataggg fwd² PCR primer to amplify library dimersrpTFT1 CGggctttgttagcagccgg rev² PCR primer to amplify library dimer¹small letters indicate regions designed for annealing ²abbreviations:fwd - forward; rev - reverse. ³NNN stands for a mixture oftrinucleotides.

TABLE 2Oligonucleotides used for the generation of ankyrin repeat modulesaccording to example 2 (SEQ ID NOS 38-43, respectively in order of appearance)Name Sequence in 5′-3′ direction (restriction sites) Description INT1CTGACGTTAACGCTNNNGACNNNNNNGGTNNNACTCCGCTGCACCTGGC ¹Forward primer (1) for the assembly of ankyrin repeat modules INT2ACTCCGCTGCACCTGGCTGCTNNNNNNGGTCACCTGGAAATCG ¹Forward primer (2) for the assembly of ankyrin repeat modules INT3AACGTCAGCACCGTDCTTCAGCAGAACTTCAACGATTTCCAGGTGACC ²Reverse primer (1) for the assembly of ankyrin repeat modules INT4AGCAGCCAGGTGCAGCGGAGT Reverse primer (2) for the assembly ofankyrin repeat modules INT5TTCCGCGGATCCTAGGAAGACCTGACGTTAACGCT(BamHI, BpiI)Forward primer for ankyrin repeat moduleand C-terminal ankyrin capping module amplification (BpiI) INT6aTTTGGGAAGCTTCTAAGGTCTCACGTCAGCACCGT (HindIII, BsaI)Reverse primer for ankyrin repeat moduleand N-terminal ankyrin capping module amplification (BsaI) ¹ NNN standsfor a mixture of trinucleotides encoding the amino acids A, D, E, F, H,I, K, L, M, N, Q, R, S, T, V, W and Y (Vimekãs et al., 1994). ² Drepresents A, T or G.

TABLE 3Oligonucleotides used for the generation of the N- and C-terminal ankyrin capping modulesas well as for the cloning of ankyrin repeat proteins containing more than oneankyrin repeat module (SEQ ID NOS 44-52, respectively in order of appearance)Name Sequence in 5′-3′ direction (restriction sites) Description INT6bTTTGGGAAGCTTCTAAGGTCTC(HindIII, BsaI)Reverse primer for the amplification ofankyrin repeat modules having a INT6a sequence at the 3′ end INT6TTTGGGAAGCTTCTAGAAGACAACGTCAGCACCGT (HindIII, BpiI)Reverse primer for amplification of the N-terminal ankyrin capping module (BpiI) EWT1TTCCGCGGATCCGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTForward primer for the assembly of the N- CAGGACGACGAAGterminal ankyrin capping module EWT2AACGTCAGCACCGTTAGCCATCAGGATACGAACTTCGTCGTCCTGACCReverse primer for the assembly of the N-terminal ankyrin capping module EWT3 TTCCGCGGATCCGACCTGGG (BamHI)Forward primer (1) for the amplification ofsequences containing the N-terminal ankynn capping module TEN3TTCCGCGGATCCG (BamHI) Forward primer (2) for the amplification ofsequences containing the N-terminal ankyrin capping module WTC1CTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCAForward primer for the assembly of the C- TCGACAACGGTAACGAGGterminal ankyrin capping module WTC2TTGCAGGATTTCAGCCAGGTCCTCGTTACCGTTGTCReverse primer for the assembly of the C-terminal ankyrin capping module WTC3TTTGGGAAGCTTCTATTGCAGGATTTCAGC (HindIII)Reverse primer (1) for the amplification ofsequences containing the C-terminal ankyrin capping module

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The invention claimed is:
 1. A method for the construction of acollection of nucleic acid molecules, comprising the steps of (a)identifying a repeat unit from a repeat protein family; (b) identifyingframework residues and target interaction residues in said repeat unit;(c) deducing a repeat module comprising fixed positions and randomizedpositions from at least one member of said repeat protein family; and(d) constructing nucleic acid molecules each encoding a repeat proteincomprising a repeat domain, wherein said repeat domain comprises two ormore copies of said repeat module deduced in step (c), wherein thecollection of nucleic acid molecules encodes a collection of repeatproteins, and wherein said repeat proteins differ from each other in atleast one position of said two or more copies of said repeat modulededuced in step (c).
 2. The method of claim 1, wherein said at least onerepeat module deduced in step (c) has an amino acid sequence, wherein atleast 70% of the amino acid residues correspond either (i) to consensusamino acid residues deduced from the amino acid residues found at thecorresponding positions of at least two naturally occurring repeatunits; or (ii) to the amino acid residues found at the correspondingpositions in a naturally occurring repeat unit.
 3. A method for theproduction of a collection of repeat proteins, comprising the steps of(a) providing a collection of host cells comprising the collection ofnucleic acid molecules produced by the method of claim 1, and (b)expressing said collection of nucleic acid molecules comprised in saidhost cells.
 4. A method for obtaining a repeat protein having apredetermined property, comprising the steps of (a) providing thecollection of repeat proteins produced by the method of claim 3, and (b)screening said collection and/or selecting from said collection ofrepeat proteins to obtain at least one repeat protein having saidpredetermined property.
 5. The method of claim 4, wherein saidpredetermined property is activation of a target-mediated reaction. 6.The method of claim 4, wherein said predetermined property is enzymaticactivity.
 7. The method of claim 4, wherein said predetermined propertyis binding to a target.
 8. The method of claim 7, wherein the target isa nucleic acid molecule.
 9. The method of claim 7, wherein the target isa (poly)peptide or protein.
 10. The method of claim 7, wherein thetarget is a carbohydrate.
 11. The method of claim 7, wherein the targetis a whole cell.
 12. The method of claim 7, wherein the target is atissue sample.
 13. The method of claim 1, wherein said repeat unit is anankyrin repeat.
 14. A method for the construction of a collection ofnucleic acid molecules, comprising the steps of (a) selecting at leastone repeat module comprising fixed positions and randomized positions,wherein said at least one repeat module is derived from a repeat unit ofat least one member of a repeat protein family; and (b) constructingnucleic acid molecules each encoding a repeat protein comprising arepeat domain, wherein said repeat domain comprises two or more copiesof said at least one repeat module selected in step (a), wherein thecollection of nucleic acid molecules encodes a collection of repeatproteins, and wherein said collection of repeat proteins comprisesrepeat proteins that differ from each other in at least one position ofsaid two or more copies of said at least one repeat module selected instep (a).
 15. The method of claim 14, wherein said at least one repeatmodule selected in step (a) has an amino acid sequence, wherein at least70% of the amino acid residues of said amino acid sequence correspondeither (i) to consensus amino acid residues deduced from amino acidresidues found at corresponding positions of at least two naturallyoccurring repeat units; or (ii) to amino acid residues found atcorresponding positions in a naturally occurring repeat unit.
 16. Themethod of claim 14, wherein said repeat unit is an ankyrin repeat. 17.The method of claim 16, wherein said at least one repeat modulecomprises the ankyrin repeat consensus sequenceDxxGxTPLHLAaxx±±±±±±±±±±GpxpaVpxLLpxGA±±±±±DVNAx (SEQ ID NO: 1), wherein“x” denotes any amino acid, “±” denotes any amino acid or a deletion,“a” denotes an amino acid with an apolar side chain, and “p” denotes aresidue with a polar sidechain.
 18. A method for the production of acollection of repeat proteins, comprising the steps of (a) providing acollection of host cells comprising the collection of nucleic acidmolecules produced by the method of claim 14, and (b) expressing thesaid collection of nucleic acid molecules comprised in said host cells.19. A method for obtaining a repeat protein having a predeterminedproperty, comprising the steps of (a) providing the collection of repeatproteins produced by the method of claim 18, and (b) screening saidcollection and/or selecting from said collection of repeat proteins toobtain at least one repeat protein having said predetermined property.20. The method of claim 19, wherein said predetermined property isbinding to a target.
 21. The method of claim 20, wherein the target is anucleic acid molecule, a (poly)peptide, a protein, a carbohydrate, awhole cell, or a tissue sample.
 22. The method of claim 20, wherein thetarget is a (poly)peptide or protein.